Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis.

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3.

Combination therapy with interferon-alpha (IFN-alpha) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20-30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1-407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 +/- 13 vs 69.4 +/- 22; P < 0.01). Subsequently, the effect of IFN-alpha plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection.

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen.

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.

Impact of an antiemetic protocol on postoperative nausea and vomiting in children.

The objective of the study was to demonstrate a decreased incidence of postoperative nausea and vomiting (PONV) in children through the use of an antiemetic protocol. PONV was recorded in children (1.5-15 years) after inpatient surgery under general anaesthesia in a prospective, interview based survey. Group 1 consisted of children having surgery 1 month before the introduction of a formalized antiemetic protocol and group 2, 2 months after its introduction. Data were collected over a 1-month period in each group. Outcome measures of nausea, emesis, antiemetic requirement and patient satisfaction were monitored for the first 24-h postoperative period. There were 272 children enrolled: 138 in group 1 and 134 in group 2. There was a difference between the two groups for gender (P=0.03), type of surgery (P=0.017), perioperative opioid (P=0.003) and perioperative antiemetic use (P=0.024). However, multivariate analysis did not demonstrate an impact on outcome from these factors. The incidence of postoperative nausea (PON) and postoperative vomiting (POV) following the introduction of the protocol was 36% and 34%, respectively. Moderate to severe nausea was decreased after introduction of the protocol (18% versus 9%, P=0.028) but moderate to severe vomiting failed to reach significance (19% versus 11%, P=0.078). The proportion of children who had repeated nausea decreased after the introduction of the protocol (17% versus 8%, P=0.02) but repeated episodes of vomiting remained unchanged (19% versus 14%). This was attributed to a significant increase in antiemetic prescribing by protocol in group 2 (10% versus 59%, P < 0.001). Patient satisfaction was high in both groups (85% versus 90%). The introduction of a postoperative antiemetic protocol improved prescribing frequency. This resulted in a decreased incidence of moderate to severe PON and a reduction in the number of patients with repeated nausea.

Epidemiologic and molecular investigation of outbreaks of hepatitis C virus infection on a pediatric oncology service.

BACKGROUND: Despite screening of blood donors, hepatitis C virus (HCV) infection can occur in patients who receive multiple transfusions. OBJECTIVE: To clarify mechanisms of nosocomial transmission of HCV. DESIGN: Epidemiologic and molecular analyses of hepatitis C outbreaks. SETTING: Pediatric oncology ward. PATIENTS: Children with cancer. MEASUREMENTS: Epidemiologic analysis, HCV RNA detection, genotyping, and hypervariable region 1 (HVR1) sequencing. RESULTS: Ten cases of infection with acute HCV genotype 3a occurred between 1990 and 1993. Sequencing of HVR1 revealed three related strains. Despite an overhaul of hygiene procedures, a patient infected with genotype 1b generated nine subsequent infected patients in 1994. Several patients had high virus titers and strongly delayed anti-HCV antibody responses. All had permanent intravenous catheters. Multidose vials used for flushing or treatment had probably been contaminated during periods of overlapping treatment. CONCLUSIONS: Contamination of multidose vials was the most likely mode of HCV transmission; therefore, use of such vials should be restricted. Rigorous adherence to hygiene routines remains essential to preventing transmission of bloodborne infections.

Rapid isolation of phage displayed antibodies to beta-actin eluted from two-dimensional electrophoresis gel.

We describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two-dimensional gel electrophoresis (2-DE), using beta-actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng beta-actin per cycle, approximately 8 X 10(3) phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody-positive clones produced antibody fragments reacting against beta-actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC-CDR3-region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.

Phage-displayed Fab fragments against anti-human interleukin-2 receptor alpha. Detection of antigen-bound phages with anti-cpIII monoclonal antibodies.

The genes encoding the VHCH1 and VLCL parts of the mouse anti-human IL-2R alpha antibody 7G7B6 were amplified by PCR and the corresponding antibody fragments displayed on the surface of filamentous phages. The expression of Fab fragments was analysed by immunoblotting using HRP-labelled goat anti-mouse Ig antisera. By traditional hybridoma technology, splenocytes from Balb/c mice, immunized with native phage particles, were fused with P3X63-Ag8.653 myeloma cells in order to yield monoclonal antibodies against filamentous phage proteins. The obtained monoclonal antibody IF8 (mu/kappa) recognized the minor coat protein III as a 65-70 kDa protein band by immunoblotting, whereas the monoclonal antibody IVC8 (mu/kappa), in addition to cpIII, recognized a protein with an approximate molecular weight of 38-43 kDa. Both antibodies were employed to determine the binding specificity of the phage-displayed anti-human IL-2R alpha Fab fragments in an ELISA using recombinant baculovirus-expressed human IL-2R alpha proteins as antigens.

Patients infected with the same hepatitis C virus strain display different kinetics of the isolate-specific antibody response.

The antibody response to the hypervariable region of the E2 protein (HVR1) of hepatitis C virus (HCV) was studied in 5 patients who were infected by a common virus strain during an outbreak in a hemodialysis unit. Two patients resolved the infection, while 3 developed chronic HCV infection. For studying the antibody response to HVR1 during the early phase of infection, a Western blot assay using recombinant phage displaying HVR1 was developed. The 2 patients with resolving infection had a more rapid antibody response to HVR1 than did the patients developing chronic infection. Anti-HVR1 antibodies were repeatedly absent in 1 of the chronically infected patients. Antibodies to recombinant E2 protein occurred later than the anti-HVR1 antibodies and did not correlate with resolution of the infection. Thus, the present results suggest that early appearance of antibodies to the HVR1 may predict clearance of HCV infection.

Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells.

Human antibodies against HIV-1 have been sought to study neutralization events on the molecular level, and for possible use in passive immune intervention. The development of phage display techniques has opened the possibility of rapidly generating human monoclonal antibodies with desired specificities. We and others have isolated human HIV-1 neutralizing antibody fragments using this technique. Bacterial expression of isolated clones does, however, differ broadly both in expression levels and functional activity. In addition, intact IgG cannot be expressed in bacteria. By transferring the genes of isolated Fab clones to a mammalian expression system we could perform a comparison of functional activity between Fab expressed in bacterial and mammalian cells, as well as Fab and whole IgG. Fab fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in Escherichia coli, underlining the inefficiency of procaryotic expression. No difference in HIV-1 neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells. Thus, bivalency does not always confer improved neutralization efficacy.

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV-1 envelope protein gp120LAI, and has previously been shown to interact with the -I-GPGRA- motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif -GPGRA-, a similar segment, -GPAR-, and two unrelated motifs -FRLLG- and -WRM/ALG- were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide-specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the -GPGRA- containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV-1SF2 virus in vitro.

Frequent patient-to-patient transmission of hepatitis C virus in a haematology ward.

Blood transfusion is a well-documented route of transmission of hepatitis C virus (HCV). However, a persisting high frequency of HCV infections was recorded in our haematology ward even after screening of blood donors had been introduced. We investigated the viral strains in 37 patients with haematological malignant diseases who had developed hepatitis C when treated in the ward during 1990-93. 17 of the patients acquired hepatitis C despite being transfused only with blood components screened by second-generation anti-HCV tests. The viral strains were characterised by PCR genotyping and nucleotide sequencing of the hypervariable region of the E2 gene. Five clusters of closely related or identical viruses were found involving 2, 3, 4, 6, and 15 patients, respectively. Blood components could be ruled out as the common source of infection because no donor had given blood to all patients sharing a specific strain, and even donors whose blood had been given to several patients were negative for HCV RNA. All patients in each cluster had been treated in the ward during overlapping periods. These findings suggest that despite strict hygienic control, HCV transmission occurred between patients treated in the same hospital setting, as has previously been reported in a smaller group of haemodialysis patients.

Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus.

A collection of simian immunodeficiency virus (SIV) neutralizing recombinant Fab fragments was generated using the combinatorial antibody library approach. Functional antibody fragments efficiently expressed in Escherichia coli were identified only in the form of chimeric macaque heavy chain gamma 1 and human light chain kappa. The gamma 1 and kappa chains were derived from a clinically healthy long-term surviving SIVsm-infected cynomolgus macaque and from an asymptomatic HIV-2 seropositive individual, respectively. The combinatorial library was constructed on the surface of filamentous phage using the pComb3 phagemid vector and screened against purified SIVsm surface glycoprotein (gp148). Twelve chimeric clones reacting with the antigen were isolated. Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 micrograms/ml. All neutralizing Fab fragments were clonally unrelated as demonstrated by nucleic acid sequencing. These potent neutralizing reagents will be used for prophylactic and therapeutic immune intervention of lentivirus infection in macaques and to map neutralizing determinants of SIV.

Hepatitis C transmission in a hemodialysis unit: molecular evidence for spread of virus among patients not sharing equipment.

Three cases of simultaneous seroconversion to hepatitis C virus (HCV) in a hemodialysis unit initiated the investigation of the viral strains of 14 seropositive patients in the unit by nucleotide sequencing. The results showed that five patients had been infected with the same viral strain, and indicated that two other patients were sharing a second strain. Transmission was not related to blood transfusions and not associated with the dialysis machines, but occurred between patients treated on the same shift. The number of cases was higher than expected from the serological data. Thus, spread of virus may occur at high frequencies in environments where parenteral routes are made accessible, in spite of rigorous preventive measures. This may raise concern that non-transfusion associated spread of HCV may be present and unnoticed in several hospital settings.

Combinatorial libraries.

Combinatorial antibody libraries, in which PCR amplified immunoglobulin light and heavy chain DNA are randomly recombined irrespective of their pairing in vivo into a vector and subsequently expressed in E. coli, have quickly become a very productive tool to generate monoclonal antibodies from various species. It has been drastically improved by utilizing phage display technologies in the selection process of specific antibodies. A brief summary of current techniques, critical published experiments showing the versatility of these systems with emphasis on human antibodies and discussions on chain preference, affinity maturation and the advent of semisynthetic and non-immune libraries will be presented.

[A new genetic technology makes the production of human monoclonal antibodies simpler]. / Nya genteknologier förenklar framställningen av humana monoklonala antikroppar.

New advances in DNA technology have enabled repertoires of antibodies to be cloned and expressed in bacteria (E coli), thus creating libraries of antibody DNA. In principle, heavy and light chain DNA is amplified from lymphocyte RNA and combined into vectors that enable antibody molecules to be expressed on the surface of phage particles which carry the DNA for the corresponding antibody in their genomes. The system constitutes an ideal tool for use in selecting even rare antibodies from libraries of millions. The new advances have greatly facilitated the generation of monoclonal antibodies against various substances. In particular, human monoclonal antibodies are now more easily obtained with DNA technology than with the methods previously used. Many of the antibodies generated with the new techniques were derived from lymphocyte RNA from immune donors, though the isolation of specific antibodies from libraries originating from donors who have never been in contact with the particular antigens has recently been reported. Moreover, the binding characteristics of the immunoglobulins isolated have subsequently been enhanced in vitro. Thus, modern antibody cloning techniques give promise of allowing the generation of a wide range of specific antibodies from a single, diverse library of antibodies kept 'on the shelf' in the laboratory, without needing to immunise humans or animals. The impact on biomedical science is likely to be profound, as specific reagents for use in research or therapy will be easily obtained, irrespective of whether human or other mammalian antibodies are required.

Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies.

A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals.

A panel of human monoclonal antibody Fab fragments has been generated against the surface glycoprotein gp120 of type 1 human immunodeficiency virus (HIV) by antigen selection from a random combinatorial library expressed on the surface of filamentous phage. The library was prepared from 5 ml of bone marrow from an asymptomatic individual who has been HIV-positive for 6 years. The antibodies have high affinity for antigen (mostly with affinity constants of greater than 10(8) M-1) and notable sequence diversity. Given appropriate donor selection, the methods described should allow the generation of antibodies for the evaluation of passive immunization as a therapy for AIDS.

Expression of a human monoclonal anti-(rhesus D) Fab fragment in Escherichia coli with the use of bacteriophage lambda vectors.

A human anti-(rhesus D) antibody (IgG1 lambda) Fab fragment was cloned from an Epstein-Barr-virus-transformed cell line and expressed in Escherichia coli with the use of bacteriophage lambda vectors. The cloned protein is active in binding to human erythrocytes and permits the development of a recombinant reagent for the prevention of haemolytic disease of the newborn. The method offers a rapid and effective means of rescuing human Fabs from potentially unstable cell lines secreting human antibodies.