Elevated plasma high-mobility group box 1 protein is a potential marker for neuromyelitis optica.

High-mobility group box 1 protein (HMGB1) has cytokine activities and mediates systemic inflammation as well as immune responses. The aim of this study was to determine if plasma HMGB1 level can be used as a marker for neuromyelitis optica (NMO) and to differentiate NMO from multiple sclerosis (MS). We measured plasma levels of HMGB1, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin 17 (IL-17) in 29 patients with NMO and 20 patients with MS at enrollment and at 2years follow-up (at the time of definitive diagnosis) by enzyme-linked immunosorbent assay. Plasma HMGB1 level was significantly greater in the NMO group compared to the MS group (P<0.001). Plasma levels of TNF-α, IFN-γ, and IL-17 were significantly greater in the NMO group compared to the MS group, and HMGB1 level was positively correlated with TNF-α, IFN-γ, and IL-17 levels. Univariate logistic regression analysis showed a significant association of HMGB1 level, and IFN-γ level with NMO diagnosis. Although this study included a limited sample size, we attempted to determine an optimized cutoff point for HMGB1 (≥2 ng/ml), which provided 89.7% sensitivity and 95.0% specificity for the diagnosis of NMO. These results indicate that plasma HMGB1 level might serve as a surrogate marker for NMO disease activity and aid in the differentiation of NMO from MS at the early disease stage.

The impact of donor variables on the outcome of orthotopic liver transplantation for hepatitis C.

Morphologic characteristics of the graft have been proposed as a major contributor to the long-term outcomes in orthotopic liver transplantation (OLT). Our objective was to determine the impact of donor variables, including donor age, donor-recipient HLA match, and type of donation (DCD vs donation after brain death [DBD]), on the outcome of OLT in 192 patients with hepatitis C virus (HCV). Fourteen patients underwent OLT from donation after cardiac death (DCD) donors and 188 from DBD donors. Mean donor age, warm ischemia time at recovery, and cold ischemia time were similar between the groups. Overall graft survival rate at 1 year (55% DCD vs 85% DBD) and 5 years (46% DCD vs 78% DBD) was significantly lower in the DCD group (P = .0003). Similarly, patient survival rate at 1 year (62% DCD vs 93% DBD) and 5 years (62% DCD vs 82% DBD) was significantly lower in the DCD group (P = .0295). Incidences of hepatic artery thrombosis, portal vein thrombosis, and primary nonfunction were similar between the DCD and DBD groups. The incidence of liver abscess with ischemic-type biliary stricture was higher in recipients from DCD as compared with DBD (42% vs 2%). A trend toward lower graft survival was noted in recipients from donors older than 60 years of age in the HCV population (P = .07), with statistically lower patient survival (P = .02). Donor- recipient HLA matching did not appear to correlate with OLT outcome in patients with HCV. DCD donors and donors older than 60 years of age significantly impact patient and graft survival. Lower graft and patient survival in recipients from DCD donors does not appear to be related to early disease recurrence.

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, 2,311 (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.

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.

Living-unrelated renal transplantation at the University of Wisconsin.

Since 1984, we have performed 243 living-unrelated renal transplants at the University of Wisconsin. Rejection occurred in 47% of the patients. Graft loss occurred in 59 patients and 39 patients died. Graft survival in LURD transplants at 10 years is 54% and 43% at 15 years. Patient survival is 68% at 10 years and 54% at 15 years. These long-term results demonstrate that LURD is equivalent to haploidentical renal transplantation and superior to cadaveric transplantation. Husband-to-wife donation demonstrated improved graft survival when compared with wife-to-husband and nonspousal donation. Living-unrelated renal transplantation has been utilized successfully at the University of Wisconsin and may help to alleviate the donor shortage.

Seroprevalence of CMV antibodies in a blood donor population and premature neonates 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.

In vitro immunization of naive human B cells yields high affinity immunoglobulin G antibodies as illustrated by phage display.

In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen-specific isotype switch, which was dependent on the presence of antigen-specific T-helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti-V3-specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen-specific Fab. representing different phases of the humoral in vitro immune response were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen-specific in vitro immune response can yield high-affinity immunoglobulinG antibodies.

Evaluation of screening kits for the detection of anti-human immunodeficiency virus type 1 and 2 (HIV-1/2) antibodies.

HIV-1/HIV-2 3rd generation (Abbott), Wellcozyme HIV 1 + 2 (Murex), Enzygnost Anti-HIV 1/-HIV 2 (Behring), and Genelavia Mixt (Sanofi Diagnostics Pasteur) are currently registered by authorities as enzyme immunoassays (EIAs) for detecting HIV-1/2 infection. The present study dissects these reagents by means of the major antigenic components, assay principles and their actual performance. The performances have been evaluated by their test results in international panels of seroconversion, mixed titer performance and HIV-1/2 combination, respectively. Those EIA tests were further used to examine 26 potentially false-reacting samples, serial diluted sera prepared from two confirmed positive specimens and 720 specimens obtained from random blood donors in the Taipei Blood Center, Chinese Blood Services Foundation (CBSF). The results showed that, although standard sera of the mixed titer, performance and HIV-1/2 combination rows could not distinguish significantly among various EIAs, the seroconverting samples clearly showed their differences. The differences, as calculated by using 3 of 4 seroconverting sera, was a backward window period ranging from 19 to 23 days as compared to the detection of HIV-1 antigens. Together, these studies strongly suggest that assays which are capable of detecting HIV-specific IgM and IgG antibodies have a shorter seroconversion window. Furthermore, the HIV-2 antigen seems to be crucial for successful detection of anti-HIV-2. Finally, testing anti-HIV-1/2 in the routine screenings is expected not to increase the exclusion rate of blood units currently acquired from the examination of anti-HIV-1. Consequently, with both HIV-1/2 specificities and the ability of early detection, IgM/IgG-captured EIAs may represent a better screening method than assays based solely on the detection of HIV-specific IgG.

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors.

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4+ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 microgram/ml.

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.

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 autologous Epstein-Barr virus (EBV)-transformed lymphoblastoid cells; (ii) a phenotype characteristic for the helper/inducer CD4+/CD8-/CD450R0+ 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.

Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors.

The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T-cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287-467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significantly frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes, mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.

CD4 and CD8 act as co-receptors during thymic selection of the T cell repertoire.

The selection of T cell receptor specificities must logically not only involve the alpha beta-TCR but, also the CD4 and CD8 molecules, as antigen recognition by the alpha beta-TCR on mature T cells is facilitated by the CD4 and CD8 co-receptors. In this review, the studies that provided key advances in our understanding of the possible role of CD4 and CD8 in T cell development will be discussed.

Peripheral clonal elimination of functional T cells.

A major mechanism for generating tolerance in developing T cells is the intrathymic clonal deletion of T cells that have receptors for those self antigens that are presented on hematopoietic cells. The mechanisms of tolerance induction to antigens not expressed in the thymus remain unclear. Tolerance to self antigens can be generated extrathymically through the induction of clonal nonresponsiveness in T cells with self-reactive receptors. A second mechanism of extrathymic tolerance was identified: clonal elimination of mature T cells with self-reactive receptors that had previously displayed functional reactivity.

Failure of clonal deletion in neonatally thymectomized mice: tolerance is preserved through clonal anergy.

Self-tolerance is achieved in part through intrathymic deletion of self-reactive T cells. The necessity of the thymus for this process is suggested by the development of autoimmune diseases in neonatally thymectomized (neoTx) mice and by the failure of clonal deletion in nude mice. Indeed, the present study demonstrates that neonatal thymectomy on day 3 after birth results in the failure of clonal deletion of V beta 11+ T cells in BALB/c mice and V beta 5+ and V beta 6+ T cells in DBA/2 mice. However, these potentially autoreactive cells are nonfunctional as measured by proliferation and lymphokine production after stimulation with appropriate anti-V beta mAbs or stimulator cells. It appears that this induction of nonresponsiveness may have occurred extrathymically: the early neonatal thymus (presumably the source of the peripheral T cells observed in neoTx mice) also contains T cells with self-reactive receptors, but these cells are fully functional. Therefore, neonatal thymectomy aborts deletion of self-reactive T cells, but self-tolerance is maintained through functional inactivation of potentially self-reactive clones.

Acquisition of self-tolerance in T cells is achieved by different mechanisms, operating both inside and outside the thymus.

During the past 3 years, considerable progress has been generated in understanding the mechanisms by which developing T cells acquire tolerance for self-antigens. Clonal deletion of T cells with self-reactive receptors for certain antigens expressed in the thymus was the first process to be identified as a major mechanism for generating tolerance in thymocytes. When this process fails, nondeletional mechanisms for acquisition of tolerance to self-antigens are employed. If the molecular mechanisms underlying these events are to be understood, it is crucial to identify to which extent these alternate forms of tolerance induction operate at different stages of T cells development. This article reviews where and how the tolerant state is achieved at different stages of T cell development.

Comparison of three related methods to select T cell-presented sequences of protein antigens.

A comparison of three methods to predict T cell-presented sequences within antigenic proteins led to the view that recurrent hydrophobic residues might nucleate excised peptides as alpha-helices against hydrophobic surfaces. Such helices could be protease-protected structures on their way to desetope binding. The compared methods were: the amphipathicity algorithm of DeLisi and Berzofsky [Proc. natn. Acad. Sci. U.S.A. 82, 7048-7052. (1985)] as modified by Margalit et al. [J. Immun. 138, 2213-2229. (1987)] the strip of-helix hydrophobicity algorithm (SOHHA) of Stille et al. [Molec. Immun. 24, 1021-1027. (1987)] and the motifs algorithm of Rothbard and Taylor [EMBO J. 7, 93-100. (1988)]. Correct prediction was defined at two levels of stringency: (1) the predicted sequence overlapped the experimentally reported sequence when the ratio of the intersection of both to the union of both greater than or equal to 0.5 or (2) the sequences touched when there was a non-empty intersection of both sequences. We determined the sensitivity (correct predictions/number of reported T cell-presented sequences) and efficiency (correct predictions/number of predictions) at each level of stringency. In terms of overlap, the SOHHA was more sensitive (0.43) than the amphipathicity (0.29) (not significant) and motifs (0.0, 0.0) (p less than 0.05) predictions and more efficient (0.35) than the amphipathicity (0.14) and motifs (0.0, 0.0) predictions. At the less stringent criterion touching, the amphipathicity method (0.71) was as sensitive as motif Rothbard-4 (0.79) and more sensitive than SOHHA (0.57) and motif Rothbard-5 (0.43). At that criterion, the SOHHA was more efficient (0.47) than the amphipathicity (0.36) and motifs (0.25, 0.40) methods. We hypothesize that the comparability of these approaches reflected the common, predominant influence of recurrent hydrophobicity in their predictions.

Characterization of four newly established human colorectal adenocarcinoma cell lines from Chinese patients.

Four colon adenocarcinoma cell lines, CC-M2, CC-M3, CC-M4, and CC-M2NM, have been established from surgical specimens of 18 unselected patients without the use of "feeder" cells and additional growth factors (e.g., insulin, hydrocortisone, etc.) in the culture medium. The methods of primary cultivation of tissue explants are described. Studies of determination of morphology, growth curve, plating efficiency, chromosomal analysis, CEA and beta-HCG synthesis, and tumorigenicity, were done to characterize the cell lines. Significant variations have been found in one of the four cell lines, both in vitro and in vivo studies. There are distinct phenotypes in the established cell lines which may be useful in studying the cell differentiation and progression of colorectal cancer.

Selection of class I MHC-restricted peptides with the strip-of-helix hydrophobicity algorithm.

A strip-of-helix hydrophobicity algorithm to predict class II MHC-restricted peptides, on the basis of their structural similarity to an amphipathic, alpha-helix in Ii, also predicted peptides which were presented to cytotoxic T-cells by class I MHC molecules. This algorithm ranked peptides according to mean Kyte-Doolittle hydrophobicity values of amino acids at positions n, n + 4, n + 7, n + 11, n + 14 and n + 18 in a sequence which when coiled as a putative alpha-helix, had the indicated residues in an axial strip along one side of the helix. Sequences selected for highly scoring, hydrophobic strips were required to have at least 1 of the 4 adjacent strips scoring more negatively than -1 in the strip-of-helix hydrophobicity index and the entire sequence could contain no prolines. This algorithm predicted the class I MHC-restricted, T-cell-presented peptides in sequences of 4 proteins from which some class I MHC-restricted, T-cell-presented sequences had been experimentally determined. Since both class I and class II MHC-restricted peptides could be identified with this algorithm, one can propose that: (1) foreign peptide-binding sites (desetopes) of the class I and class II MHC molecules are structurally similar; and (2) any one T-cell-presented peptide can be presented by some specific allele of both a class I and a class II MHC antigen.