An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients

We describe the development of a capture enzyme-linked immunosorbent assay for the detection of the dengue virus nonstructural protein NS1. The assay employs rabbit polyclonal and monoclonal antibodies as the capture and detection antibodies, respectively. Immunoaffinity-purified NS1 derived from dengue 2 virus-infected cells was used as a standard to establish a detection sensitivity of approximately 4 ng/ml for an assay employing monoclonal antibodies recognizing a dengue 2 serotype-specific epitope. A number of serotype cross-reactive monoclonal antibodies were also shown to be suitable probes for the detection of NS1 expressed by the remaining three dengue virus serotypes. Examination of clinical samples demonstrated that the assay was able to detect NS1 with minimal interference from serum components at the test dilutions routinely used, suggesting that it could form the basis of a useful additional diagnostic test for dengue virus infection. Furthermore, quantitation of NS1 levels in patient sera may prove to be a valuable surrogate marker for viremia. Surprisingly high levels of NS1, as much as 15 mu g/ml, were found in acute-phase sera taken hom some of the patients experiencing serologically confirmed dengue 2 virus secondary infections but was not detected in the convalescent sera of these patients. In contrast, NS1 could not be detected in either acute-phase or convalescent serum samples taken from patients with serologically confirmed primary infection. The presence of high levels of secreted NS1 in the sera of patients experiencing secondary dengue virus infections, and in the context of an anamnestic antibody response, suggests that NS1 may contribute significantly to the formation of the circulating immune complexes that are suspected to play an important role in the pathogenesis of severe dengue disease.

Autoimmune Neurological Disease

This book provides a comprehensive and critical overview of the immunological aspects of autoimmune neurological disease. These diseases include common conditions such as multiple sclerosis, the Guillain–Barré syndrome and myasthenia gravis. The introductory chapters on antigen recognition and self–non-self discrimination, and on neuroimmunology, are followed by chapters on specific diseases. These are presented in a standardized format with sections on clinical features, genetics, neuropathology, pathophysiology, immunology and therapy. Each chapter has a concluding section which summarizes key points and suggests directions for future research. Animal models of autoimmune neurological disease are also covered in detail because of their importance in understanding the human diseases. The book is suitable for clinicians and neurologists managing patients with these diseases, and for immunologists, neuroscientists and neurologists investigating the pathogenesis and pathophysiology of these disorders.

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus or bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses. (C) 1998 Academic Press.

Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.

Isolation of genotype- and chromosome-specific DNA markers in a biotype of Colletotrichum gloeosporioides using random amplified polymorphic DNA analysis

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype-and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies

Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coil with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens. (C) 1998 Academic Press.

Role of generalized and episode specific memories in the word frequency effect in recognition

Frequency, recency, and type of prior exposure to very low-and high-frequency words were manipulated in a 3-phase (i.e., familiarization training, study, and test) design. Increasing the frequency with which a definition for a very low-frequency word was provided during familiarization facilitated the word's recognition in both yes-no (Experiment 1) and forced-choice paradigms (Experiment 2). Recognition of very low-frequency words not accompanied by a definition during familiarization first increased, then decreased as familiarization frequency increased (Experiment I). Reasons for these differences were investigated in Experiment 3 using judgments of recency and frequency. Results suggested that prior familiarization of a very low-frequency word with its definition may allow a more adequate episodic representation of the word to be formed during a subsequent study trial. Theoretical implications of these results for current models of memory are discussed.

Vaccine-induced Th1-type responses are dominant over Th2-type responses in the short term whereas pre-existing Th2 responses are dominant in the longer term

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)(2a) antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil-A adjuvant, but not complete Freund's adjuvant (CFA) or Algammulin, induced a T-helper 1 (Th1)-type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7-specific IgG(2a). All tested adjuvants elicited comparable levels of E7-specific IgG(1). The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil-A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)-promoting adjuvant gave a Th1-type response. However, E7 and Quil-A were unable to induce a Th1-type response (as measured by the inability to generate anti-E7 IgG(2a) antibody) in animals with a pre-existing Th2-type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1-type response is sought, provided that there is no pre existing Th2-type response to the antigen.

The Australian mortality decline: cause-specific mortality 1907-1990

This review describes the changes in composition of mortality by major attributed cause during the Australian mortality decline this century. The principal categories employed were: infectious diseases, nonrheumatic cardiovascular disease, external causes, cancer,'other' causes and ill-defined conditions. The data were age-adjusted. Besides registration problems (which also affect all-cause mortality) artefacts due to changes in diagnostic designation and coding-are evident. The most obvious trends over the period are the decline in infectious disease mortality (half the decline 1907-1990 occurs before 1949), and the epidemic of circulatory disease mortality which appears to commence around 1930, peaks during the 1950s and 1960s, and declines from 1970 to 1990 (to a rate half that at the peak). Mortality for cancer remains static for females after 1907, but increases steadily for males, reaching a plateau in the mid-1980s (owing to trends in lung cancer); trends in cancers of individual sites are diverse. External cause mortality declines after 1970. The decline in total mortality to 1930 is associated with decline in infection and 'other' causes, Stagnation of mortality decline in 1930-1940 and 1946-1970 for males is a consequence of contemporaneous movements in opposite directions of infection mortality (decrease) and circulatory disease and cancer mortality (increase). In females, declines in infections and 'other' causes of death exceed the increase in circulatory disease mortality until 1960, then stability in all major causes of death to 1970. The overall mortality decline since 1970 is a consequence of a reduction in circulatory disease,'other' cause, external cause and infection mortality, despite the increase in cancer mortality (for males).

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.