bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa
Authors: Hsiang  , Mahuku
Published: March 2003

Restriction fragment length polymorphisms (RFLP) of the intergenic spacer region (IGS) of rDNA and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability among 181 isolates of Sclerotinia homoeocarpa from Ontario and 10 isolates from Japan. RAPD and IGS‐RFLP analyses revealed polymorphisms within and between populations of S. homoeocarpa, distinguishing 151 genotypes. Both types of markers gave similar results in phenetic analysis of genetic distances between populations. Cluster analysis showed that Japanese isolates of S. homoeocarpa were genetically distinct from Ontario isolates, demonstrating significant intraspecific differentiation. An average genetic similarity of 0.66 was found between Japanese isolates. Among Ontario isolates, average genetic similarity was 0.86, and genotypic diversity analysis showed that 49.3% of the total genetic variation observed within Ontario populations occurred among individuals within populations compared to 50.7% between populations. Gametic linkage disequilibrium analysis within Ontario populations revealed an average 15.6% significant nonrandom associations between putative RAPD loci, and that half of the populations showed signs of significant linkage disequilibrium. These results suggest that both clonal propagation and recombination events occurred in local populations of S. homoeocarpa. The high level of genetic similarity between populations and the low levels of intraspecific genetic variation may reflect a small founding population for southern Ontario isolates of S. homoeocarpa.
Differentiation of Seiridium species associated with virulent cankers on cypress in the Mediterraneanregion by PCR‐SSCP
Authors: Moricca S. , Børja I. , Vendramin G. G. , Raddi P.
Published: March 2003

A rapid and sensitive method was developed to discriminate between Seiridium cardinale and Seiridium cupressi, the fungi causing severe cankers on common cypress in the Mediterranean area. The method amplified sequence variants in the ITS2 region of ribosomal DNA using the polymerase chain reaction (PCR), followed by polyacrylamide gel electrophoresis, to reveal single‐strand conformation polymorphism (SSCP) between the two species. The greatest separation pattern was obtained with a gel matrix containing 7–10% formamide and 3–5% glycerol under optimized running conditions, which were found to be 30–40 V at 4–5°C for 4–8 h. Sequence homology among isolates within each of the two species caused no mobility shifts, with all isolates displaying the same migration pattern. A few base differences between S. cardinale and S. cupressi caused markedly different migration patterns, allowing differentiation of the two pathogens. Differences between these fungi at the genetic level are consistent with known data on morphological, physiological and pathogenic characteristics. SSCP analysis constitutes a rapid and easy‐to‐perform method by which to recognize and distinguish closely related organisms, and has considerable potential for use in diagnosis and taxonomy.
Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia
Authors: Venter M. , Wingfield M. J. , Coutinho T. A. , Wingfield B. D.
Published: March 2003

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus. Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis, were studied using PCR‐based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa. In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa, but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria.
Inoculation of pearl millet with the downy mildew pathogen, Sclerospora graminicola: chilling inoculum to delay zoospore release and avoid spray damage to zoospores
Authors: Jones E. S. , Breese W. A. , Shaw D. S.
Published: March 2003

Inoculation of plants by spraying with a suspension of zoospores (released from sporangia of Sclerospora graminicola) induced immediate encystment of the zoospores and drastically reduced their ability to cause disease. The incidence of infection in spray‐inoculated pearl millet seedlings was reduced from 90% when sporangia were sprayed, to less than 30% when zoospores were sprayed. Rapid encystment was observed when zoospores were sprayed from both a hand‐pumped sprayer and a compressed‐gas sprayer and was probably caused by shearing forces. Chilling suspensions of sporangia prior to spraying delayed zoospore release and was an effective method for maintaining infection potential. Disease incidence was higher when sporangia were chilled to 0·2°C rather than to 4°C. Chilling resulted in some abnormal zoospore structures being released from sporangia when suspensions were returned to 20°C. The frequency of these structures increased with increasing chilling time. For seedlings sprayed with sporangia before zoospore release, a small reduction in disease incidence was observed when chilled inoculum was used, probably due to cold disruption of zoosporogenesis. For large‐scale disease resistance screening, this reduction is outweighed by the benefit of a uniform and adequately high disease pressure that can be obtained over many hours using chilled spore suspensions.
Simple inheritance of partial resistance to leaf rust in two wheat cultivars
Authors: Kolmer J. A. , Liu J. Q.
Published: March 2003

The wheat cultivars Westphal 12A and BH1146 were characterized in previous studies as having partial resistance to the leaf rust pathogen Puccinia triticina. In the present study, genetic analysis showed that Westphal 12A has three genes that condition resistance in seedling plants, and the gene Lr34, which is optimally expressed in adult plants. The three seedling resistance genes in Westphal 12A may be new leaf‐rust resistance genes. BH1146 was shown to have the adult plant resistance genes Lr13 and Lr34. Many wheat cultivars with partial resistance to leaf rust may have Lr13 and/or Lr34, or other previously described Lr genes.
Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real‐time PCR
Authors: Cullen D. W. , Lees A. K. , Toth I. K. , Duncan J. M.
Published: March 2003

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus‐specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species‐specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C. coccodes. The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real‐time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first‐round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes.
Phytophthora infestans's 10‐year truce with Holland: a long‐term analysis of potato late‐blight epidemics in the Netherlands
Authors: Zwankhuizen M. J. , Zadoks J. C.
Published: March 2003

A sequence of 47 potato late‐blight (Phytophthora infestans) epidemics in the Netherlands, from 1950 to 1996, was analysed using agronomic and meteorological variables. The intensity of annual epidemics was characterized by an index of disease intensity (DI, 0 = absence of late blight; 4 = severe epidemic). Three periods were identified, with average DIs of 2·9, 0 and 2, respectively. Period I (1950–68) had relatively regular epidemics; period II (1969–78) was virtually blight free; and period III (1979–96) showed large variations in disease intensity. Disease‐enhancing factors were number of days with precipitation, and number of hours with temperatures between 10 and 27°C and relative humidity >90% during the growing season. Limiting factors were number of hours with temperatures >27°C, and amount of global radiation. Linear discriminant analysis of DI using the blight status of the previous year and meteorological variables correctly classified up to 40 years out of 47 (87·0%), with five out of the six incorrectly classified years falling in period III. Blight status of the previous year and number of days with precipitation were important discriminating variables.
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
Authors: Fernando  , Stewart  , Tindle  , Frazer
Published: March 2003

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 IgG2a. All tested adjuvants elicited comparable levels of E7‐specific IgG1. 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 IgG2a 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.
Immunotherapy Suppresses both Th1 and Th2 Responses by Allergen Stimulation, but Suppression of the Th2 Response is a More Important Mechanism Related to the Clinical Efficacy of Immunotherapy for Perennial Allergic Rhinitis
Authors: Tanaka  , Ohashi  , Kakinoki  , Nakai
Published: March 2003

Increased attention has recently been directed at the possibility that the clinical efficacy of immunotherapy might be elaborated by alteration of T‐cell reactivity. However, there is no general agreement among different investigators regarding the effect of immunotherapy on Th‐cell reactivity. Peripheral blood mononuclear cells (PBMCs) from 15 nonatopic subjects and 76 patients with perennial allergic rhinitis (18 untreated patients and 58 patients on immunotherapy) were cultured in the absence and in the presence of a major Dermatophagoides farinae allergen, Der f 1, and the levels of IgE, interleukin‐5 (IL‐5), interferon‐gamma (IFN‐γ) and tumor necrosis factor‐α (TNF‐α) in the culture supernatants were determined. The difference between the absence and presence of Der f 1 was calculated to consider the Der f 1‐dependent synthesis of IgE, IL‐5, IFN‐γ and TNF‐α. The levels of Der f 1‐dependent synthesis of IgE, IL‐5 and TNF‐α were significantly higher in the untreated group than in the nonatopic group, whereas Der f 1‐dependent synthesis of IFN‐γ was significantly lower in the untreated group than in the nonatopic group. Immunotherapy decreased the enhanced Der f 1‐dependent synthesis of IgE, IL‐5 and TNF‐α, and further decreased the suppressed Der f 1‐dependent synthesis of IFN‐γ as the therapy proceeded. The levels of Der f 1‐dependent synthesis of IgE and IL‐5 did not differ between nonatopic individuals and patients whose duration of immunotherapy was 10 or more years. The levels of Der f 1‐dependent synthesis of IgE and IL‐5, but not of IFN‐γ and TNF‐α, were correlated significantly with the levels of symptom scores. In addition, the levels of Der f 1‐dependent synthesis of IgE and IL‐5, but not of IFN‐γ and TNF‐α, differed significantly between good and poor responders. In conclusion, immunotherapy for perennial allergic rhinitis may possibly work via induction of tolerance or anergy of both Th1‐ and Th2 cells. However, our study is likely to support a view that the mechanisms responsible for the clinically beneficial effects of immunotherapy principally involve the tolerance of Th2‐ rather than Th1 cells. In addition, suppression of IgE synthesis is also likely to be linked to the clinical efficacy of immunotherapy for perennial allergic rhinitis.
Anti‐TNF Treatment of Baboons with Sepsis Reduces TNF‐α, IL‐6 and IL‐8, but not the Degree of Complement Activation
Authors: BENGTSSON  , REDL  , SCHLAG  , HÖGÅSEN  , GÖTZE  , MOLLNES
Published: March 2003

The activation of complement and the release of TNF‐α, IL‐6 and IL‐8 are important pathogenic factors behind organ dysfunction in sepsis. The aim of this study was to determine whether infusion of anti‐TNF antibodies alters complement activation and plasma concentrations of pro‐inflammatory cytokines at high doses of Escherichia coli. Six baboons received intravenously 2 × 109 live E. coli bacteria per kg body weight (group 1), in addition five received pretreatment with 1 mg per kg body weight anti‐TNF antibodies (group 2), and seven received 5 × 108 live E. coli bacteria per kg body weight (group 3). Two hours after the start of infusion of the bacteria, plasma concentrations of C3 activation products, C5a and the terminal SC5b‐9 complement complex were increased in groups 1 and 2 (P < 0.05), but there was no significant difference between the groups. At 2 h the levels of TNF‐α, IL‐6 and IL‐8 were lower in group 2 compared with group 1 (P < 0.05). In group 2 compared with group 1 the TNF‐α concentrations were, however, higher at 4, 8 and 24 h. The explanation for this phenomenon is probably that TNF‐α binds to the anti‐TNF antibody complex and is released slowly after it has been bound. The study showed that infusion of anti‐TNF antibodies reduced the concentrations of TNF‐α, IL‐6 and IL‐8, without any detectable influence on complement activation.
Immunoglobulin‐Secreting Cells in Cord Blood: Effects of Epstein–Barr Virus and Interleukin‐4
Authors: Gudmundsson  , Thorsteinsson  , Gudmundsson  , Haraldsson
Published: March 2003

The contribution of cord blood B lymphocytes to the immune response has been under considerable investigation. Cord blood B cells produce almost no antibodies except of the immunoglobulin (Ig)M isotype, indicating immaturity of the cells or the environment they reside in. The aim of this study was to investigate the number of circulating IgA‐, IgM‐, IgG‐, and IgE‐producing cells in cord blood in comparison to adult peripheral blood using the ELISPOT method. Moreover, we studied the effect of transformation with the Epstein–Barr virus (EBV) on the proportion of cells producing different isotypes with or without interleukin (IL)‐4. Cord blood had IgM‐producing cells circulating predominantly, but also some IgA‐ and IgG‐producing cells, whereas adult peripheral blood contained high amounts of circulating IgA‐producing cells and some IgM‐ and IgG‐producing cells. No circulating IgE‐producing cells were found in either group. Transformation by EBV caused significant expansion of IgA‐, IgM‐, and IgG‐producing cells in adult peripheral blood, but almost only of IgM‐producing cells in cord blood. A low but detectable expansion of IgA‐ and IgG‐producing cells was found. Cells producing IgE were still not found, even after EBV transformation. However, EBV transformation in the presence of IL‐4 increased the numbers of IgE‐producing cells significantly both in cord blood and adult peripheral blood. These findings indicate that cord blood contains some circulating IgA‐ and IgG‐producing cells that are expanded to some extent after EBV infection. They also indicate that cord blood B cells have a similar capacity for IgE production to adult peripheral blood B cells when appropriately stimulated.
Detection of B Cells and Proinflammatory Cytokines in Atherosclerotic Plaques of Hypercholesterolaemic Apolipoprotein E Knockout Mice
Authors: Zhou  , Hansson
Published: March 2003

Atherosclerosis, the main lethal disease in the Western world, is associated with a cellular immune response in the arterial lesions and a humoral immune response directed towards oxidized lipoproteins, certain microbes and other antigens. The local immune response is dominated by macrophages and T cells, while to date, the role of B cells in lesions has been unclear. We analysed B‐cell involvement in lesions using the apolipoprotein E knockout mouse, an experimental model that develops accelerated atherosclerosis when fed a lipid‐rich diet. Both early fatty‐streak‐type lesions and full‐blown atherosclerotic plaques of these mice contained CD22+ B cells. They accumulated predominantly in the base of lesions, where high expression levels of vascular cell adhesion molecule‐1 (VCAM‐1) were observed in other cells. Cells expressing interleukin‐6 and tumour necrosis factor‐α were also detected and IgM was abundant in this region. These data show that B cells participate in atherosclerosis in this experimental model; the data also suggest that these cells may accumulate through VCAM‐1 expression by surrounding cells and may produce antibodies and proinflammatory cytokines. These factors are likely to be important in the pathogenesis of atherosclerosis.
Shared Early Autoantibody Recognition Events in the Development of Anti‐Sm B/B' in Human Lupus
Authors: Arbuckle  , Reichlin  , Harley  , James
Published: March 2003

Many aspects of the immune maturation are uncharted. For ordinary human autoimmune systems there are no complete descriptions of the progression from an initial antigenic epitope to a maximally complex immune response. In this study we have exploited a large serial collection of human sera to investigate the development of the anti‐Sm autoimmune response in systemic lupus ertythematosus (SLE). The results suggest a similar, if not virtually identical, stepwise progression in the early humoral immune maturation of anti‐Sm. The amino acid sequence PPPGMRPP comprises the first epitope in the anti‐Sm B/B'response and its close relative, PPPGMRGP, the second. Epitopes are subsequently enlarged by the incorporation of neighbouring amino acids. The third and fourth epitopes are also recognised by an antibody in a nearly identical sequence in different lupus patients. A column absorption with PPPGMRPP demonstrates that the epitope spreading among the first four early epitopes appears to occur by the sequential generation of cross‐reactive antibodies. Unexpectedly, epitope spreading in this system occurs in a predictable fashion by involving essentially the same sequence of antigenic structures from person to person. In addition, these data support the lupus anti‐Sm antibodies originating against a single antigenic structure and, hence, strongly support a unifying mechanism in the generation of these autoantibodies.
Leishmania donovani Parasites Interact with γ/δ+ Human Peripheral Blood T Cells and Induce Susceptibility to NK Cell‐Mediated Lysis
Authors: Saha  , Chakrabarti  , Sen  , Bandyopadhyay
Published: March 2003

We recently reported that Leishmania donovani infect the human T‐cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of γ/δ+ T cells was markedly increased when in vitro generated normal human T‐cell blasts were cultured with L. donovani amastigotes. About 30% of the γ/δ+ T cells in the parasite exposed T‐cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T‐cell blasts had a reduced surface expression of HLA‐DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T‐cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell‐mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala‐azar patients tested expressed intracellular L. donovani antigens.
Monocytes Secrete Interleukin‐6 When Co‐Cultured In Vitro with Benign or Malignant Autologous Fragment Spheroids from Squamous Cell Carcinoma Patients
Authors: Heimdal  , Aarstad  , Olofsson
Published: March 2003

Biopsies from tumour and benign mucosa were removed from patients with head and neck squamous‐cell carcinoma (HNSCC), chopped into cubes and transferred to a nonadhesive culture system where in vitro fragment (F)‐spheroids were established. The F‐spheroids stabilized within 14 days of culture in vitro with epithelial cells and fibroblasts on the surface. F‐spheroids were co‐cultured with freshly isolated autologous monocytes. The monocytes of 10 of 11 patients secreted interleukin (IL)‐6 at a level similar to that of the average monocyte endotoxin‐stimulated response. Secreted IL‐1β or tumour necrosis factor‐α (TNF‐α) levels greater than 0.1 times the endotoxin‐stimulated secretion were determined in one and two of the 11 co‐culture experiments, respectively. This different monocyte response to F‐spheroids compared with endotoxin stimulation was also present at the mRNA expression level. HNSCC monocytes secreted no IL‐6 after co‐culture with autologous fibroblasts. When monocytes and F‐spheroids were cultured separated by a semipermeable membrane, the IL‐6 supernatant level was only ≈ 25% of that observed during co‐culture with direct contact. F‐spheroids secreted only trace amounts of IL‐6. In conclusion, monocytes of HNSCC patients generally secrete IL‐6, but not IL‐1β or TNF‐α, after stimulation with epithelial‐associated components of F‐spheroids upon direct contact and in part by a soluble substance.
Genes, Receptors, Signals and Responses to Lipopolysaccharide Endotoxin
Authors: Wong  , Chung  , Sultzer
Published: March 2003

C3H/HeJ inbred mice have been very useful for identifying genetic elements responsible for endotoxin‐mediated responses. Depending on the type of assays employed, Tlr‐2, Tlr‐4 and Lps/Ran have been shown to be important in lipopolysaccharide (LPS)‐mediated responses. The concept of a single LPS gene being responsible for the genetic defect found in C3H/HeJ mice should therefore be re‐examined more closely. Given the most recent discoveries, it is probable that more than one signal transduction pathway is involved. One is a CD14‐dependent pathway, the other a CD14‐independent pathway. Identification of the genetic elements involved in these pathways will be beneficial in designing therapeutic strategies for treating patients with endotoxic or septic shock.
Mannose Binding Lectin Polymorphisms are Associated With Early Age of Disease Onset and Autoimmunity in Common Variable Immunodeficiency
Authors: Mullighan  , Marshall  , Welsh
Published: March 2003

Mannose binding lectin (MBL) is an important component of the innate immune response. Low producing MBL alleles are associated with an increased risk of infection, especially when adaptive immunity is already compromized. We investigated the role of MBL polymorphism in common variable immunodeficiency (CVID), a disease of unknown aetiology characterized by defective humoral immunity, recurrent infections and highly variable clinical phenotype. Six biallelic single nucleotide polymorphisms in the MBL promoter and coding region (– 550, − 221, + 4, codons 52, 54 and 57) were haplotyped using a novel PCR‐SSP method in 163 CVID patients and 100 controls. Low producing coding alleles and promoter haplotypes were associated with early age of disease onset. The mean age of disease onset was 14.5 years in patients with low producing MBL coding alleles, compared with 25 years in patients with wild type coding regions (t‐test P = 0.002). Mean age of onset in patients with the low producing LXPA haplotype was 6.8 years, compared with 29.3 years for the HYPA haplotype (P = 0.003). The MBL + 4 Q allele was associated with autoimmune disease (P = 0.003, OR 4.4). These results suggest that MBL deficiency compounds the antibody deficiency in CVID, and reinforces the ostensible role of MBL in innate immunity. MBL deficiency may also facilitate the development of autoimmune disease in CVID.
Change of Th0 to Th1 Cell‐Cytokine Profile Following Tuberculosis Chemotherapy
Authors: Dieli  , Singh  , Spallek  , Romano  , Titone  , Sireci  , Friscia  , Di Sano  , Santini  , Salerno  , Ivanyi
Published: March 2003

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16‐kDa antigen‐reactive CD4 T‐cell clones prior to therapy resided mostly in disease‐associated body fluids and were of the Th0 (interferon (IFN)‐γ + interleukin (IL)‐4) secreting profile. In contrast, the majority of postchemotherapy CD4 T‐cell clones originated from blood and were of the IFN‐γ secreting Th1 type. However, the recognition of several peptides derived from the 16‐kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.
The Primary and Memory Immune Response to Epstein–Barr Virus Infection In Vitro is Characterized by a Divergent Production of IL‐1β/IL‐6 and IL‐10
Authors: Jabs  , Wagner  , Schlenke  , Kirchner
Published: March 2003

Reactivation of latent Epstein–Barr virus (EBV) infection is considered to exert substantial immunomodulating activities. Little is known about EBV's modulating activities on cytokine production upon primary, in contrast to reactivated, infection. Therefore, we investigated the cytokine production of latently infected EBV‐positive (EBV‐pos) adults upon in vitro EBV stimulation compared to nonimmune age‐matched EBV‐negative (EBV‐neg) donors. Production of interleukin (IL)‐1β and IL‐6 was strongly decreased in peripheral blood mononuclear cell (PBMC) cultures of EBV‐pos adults; in contrast, IL‐10 and IL‐1 receptor antagonist exhibited significantly higher levels. As expected, interferon (IFN)‐γ production was almost exclusively observed in EBV‐pos donors; it was accompanied by a significantly higher IL‐12 synthesis. Experiments employing T cell‐depleted PBMC showed similar cytokine levels between EBV‐pos and EBV‐neg individuals suggesting that reactivation of EBV‐specific memory T cells was responsible for the divergent cytokine profiles. Production of viral IL‐10 was excluded as a reason for higher IL‐10 levels in EBV‐pos individuals. In conclusion, our results do not appear to relate to any primary immunological differences between EBV‐neg and EBV‐pos adults, but show that the EBV‐specific memory response to latent EBV infection is characterized by some anti‐inflammatory effects. This might be of relevance upon reactivation of latent EBV infection in vivo and provides further evidence that EBV infection acts in an immunomodulating fashion.
Protective Effect of Plantago major L. Pectin Polysaccharide against Systemic Streptococcus pneumoniae Infection in Mice
Authors: Hetland G. , Samuelsen A. B. , Loslash, Vik M. , Paulsen B. S. , Aaberge I. S. , Groeng E.‐C. , Michaelsen T. E.
Published: March 2003

The antibacterial effect of a soluble pectin polysaccharide, PMII, isolated from the leaves of Plantago major, was examined in inbred NIH/OlaHsd and Fox Chase SCID mice experimentally infected with Streptococcus pneumoniae serotype 6B. Serotype 6B is known to give a more protracted infection when injected intraperitoneally into susceptible mice than more virulent serotypes like type 4. PMII was administered i.p. either once 3 days before challenge or once to thrice from 3 to 48 h after challenge. The number of bacteria in blood and the mouse survival rate were recorded. Pre‐challenge administration of PMII and also lipopolysaccharide (LPS), included as a control, gave a dose‐dependent protective effect against S. pneumoniae type 6B infection. However, injection of PMII after establishment of the infection in NIH/OlaHsd mice had no effect. The data demonstrate that, firstly, the polysaccharide fraction PMII from P. major protects against pneumococcal infection in mice when administered systemically prechallenge, and secondly that the protective effect is owing to stimulation of the innate and not the adaptive immune system.
Vertical HIV‐1 Transmission: Importance of Neutralizing Antibody Titer and Specificity
Authors: Bongertz V. , Costa C. I. , Veloso V. G. , Grinsztejn B. , João Filho E. C. , Calvet G. , Pilotto J. H. , Guimarães M. L. , Morgado M. G.
Published: March 2003

Neutralization analyses were carried out with plasma from 132 volunteer human immunodeficiency virus (HIV)‐1 infected women (76% pregnant, 24% with infants suspected for HIV‐1 infection) collected between 1994 and 1998, against autologous and heterologous primary‐ and the reference HIV‐1 MN isolates. A significantly lower percentage of HIV‐1 transmissions was observed after 1996, parallel to a more intense antiretroviral treatment of infected pregnant women. HIV‐1 isolation was significantly more frequent from peripheral blood mononuclear cells of mothers of infected children than mothers of uninfected children (P = 0.0065). Neutralization of autologous HIV‐1 isolates was comparable for HIV‐1 transmitters and nontransmitters' plasma, whereas neutralization of the reference isolate HIV‐1 MN was more frequent at high titers for pregnant women who did not transmit HIV to their offspring compared to pregnant women who did. Although neutralization of heterologous primary HIV‐1 isolates from HIV transmitters and non transmitters by transmitter plasma occurred with similar frequency, neutralization of isolates from transmitters was much more frequent when heterologous plasma from nontransmitters were used. Macrophage‐tropic heterologous HIV‐1 isolates were neutralized more frequently at higher titers by plasma from nontransmitters than from transmitters. The results obtained indicate that antiretroviral treatment, lack of success of HIV‐1 isolation and high titers of antibodies able to neutralize macrophage‐tropic viruses appear to be of importance for protection against HIV‐1 vertical transmission for the group of patients studied.
Genetically Manipulated Bacterial Toxin as a New Generation Mucosal Adjuvant
Authors: Yamamoto M. , Mcghee J. R. , Hagiwara Y. , Otake S. , Kiyono H.
Published: March 2003

Cholera toxin (CT) and heat‐labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co‐administered protein antigen (Ag). Both LT and CT induce B7‐2 expression on antigen‐presenting cells (APCs) for subsequent co‐stimulatory signalling to CD4+ T cells. CT directly affects CD4+ T cells activated via the TCR–CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)‐4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL‐4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4+ Th2 cells, which provided effective help for the Ag‐specific mucosal immunoglobulin (Ig)A, as well as serum IgG1, IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7‐2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4+ T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.
T‐Cell Receptor Repertoire in Human Germinal Centres
Authors: Källberg E. , Leanderson T. , Natvig J. B.
Published: March 2003

A search for an antigen‐driven expansion of T lymphocytes in the inflamed joints in rheumatoid arthritis (RA) patients have been going on for decades. We here analyzed the human germinal centre T‐cell receptor (TCR) Vβ gene usage with polymerase chain reaction (PCR) combined with sequence analysis, to address the question of clonality in tonsils and synovial tissue from RA patients. Our data show a large degree of TCR heterogeneity in both these histological structures. Furthermore, clonally related T cells were found within different closely located germinal centres indicating either an active T‐cell migration between germinal centres (GC) or that a T‐cell clone may seed more than one GC.
A Role for Complement C5 in Organism Containment and Granulomatous Response during Murine Tuberculosis
Authors: Actor J. K. , Breij E. , Wetsel R. A. , Hoffmann H. , Hunter Jr R. L. , Jagannath C.
Published: March 2003

The molecular mechanisms underlying protective granuloma formation and control of bacterial growth during infection with Mycobacterium tuberculosis (MTB) are not yet completely understood. MTB‐infected mice with natural deficiency in complement component C5 are unable to develop productive granulomatous responses, and are impaired in limiting organism growth within the lung. To address the molecular basis for this histologic dysfunction, congenic complement C5‐sufficient (B10.D2‐H2d H2‐T18c Hcl/nSnJ) and complement C5‐deficient strains (B10.D2‐H2d H2‐T18c Hco/oSnJ) congenic mice were infected with Mycobacterium tuberculosis, and cytokine and chemokine responses were examined. Twelve and 28 days after infection, lungs showed elevated messages for multiple inflammatory cytokines in both congenic strains. Interleukin (IL)‐12(p40) mRNA was also induced during infection in C5‐deficient mice, although levels were significantly decreased compared to C5‐sufficient congenics. C5‐deficient mice also demonstrated reduced KC, MIP‐2, IP‐10, and MCP‐1 mRNA. The defect may directly involve C5‐mediated effects on macrophage responses; C5‐deficient bone marrow derived macrophages had significantly reduced secretion of KC, MIP‐1α and MIP‐2 compared to C5‐sufficient macrophages following in vitro infection. These findings indicate a role for C5 in mediation of chemotactic and activation events that are the basis for granulomatous responses during murine tuberculosis.
A Signal Sequence Trap Based on Cell Enrichment Using Anti‐CD19 Antibody Coated Magnetic Beads
Authors: Rian E. , Wabakken T. , Hauge H. , Aasheim H.‐C.
Published: March 2003

Precursors of many secreted and cell surface proteins contain NH2‐terminal signal sequences that lead proteins into the endoplasmic reticulum and to the cell surface. Methods have been developed to clone and identify such proteins by trapping their NH2‐terminal signal sequences, so called signal sequence traps. In this study we present an alternative and simplified signal sequence trap based on the combination of a novel vector construct expressing a cDNA library in fusion with a CD19 reporter gene, transfection in mammalian cells and selection of cells expressing trapped signal sequences using magnetic beads. Using this method we have isolated several known and novel factors with signal peptides.
Segregation of Effector Mechanisms in a Tumour‐Specific CD8+ T‐Cell Clone Correlates with CD30 Expression
Authors: Sun Y. , Song M. , Maeurer M. J. , Schadendorf D.
Published: March 2003

In this study we have analyzed CD30‐antigen expression in three melanoma‐directed cytotoxic T lymphocyte (CTL) clones with a T helper 0 (Th0)‐like cytokine secretion profile (i.e. interleukin (IL)‐4, IL‐5, and interferon (IFN)‐γ). We show that all CTL clones expressed high levels of CD30 upon contact with the autologous tumour cells. One CTL clone, termed A2 with a monoclonal feature was selected for further analyses and found its CD30 expression dependent on the presence of IL‐4. Functionally, a CD30‐expressing A2 CTL was capable of producing higher amounts of IFN‐γ (up to 1.5‐fold) and IL‐4 (up to two‐fold) than its CD30− counterpart. Furthermore, CD30‐positive A2 CTL displayed an at least three‐fold greater proliferative response to the tumour cell stimulation, contrasting with CD30− CTL. However, the antitumour cytotoxic activity of A2 CTL was not modulated by the CD30 expression. These results suggest that CD30 antigen can be inducible on a subset of tumour‐directed CD8+ CTL, and that this subset of cells may have profound effector functions, such as cytokine secretion, proliferation, and cytotoxicity.
Analysis of Ig κ Light Chain Gene Variable Regions Expressed in the Rheumatoid Synovial B Cells
Authors: Pyon H. S. , Ha‐Lee Y. M. , Song G. G. , Sohn J.
Published: March 2003

Sequence analysis of antibody variable (V) regions can provide an insight regarding whether B cells have gone through an antigen‐driven process of affinity maturation. In this study, we analyzed 16 V‐regions of immunoglobulin (Ig) κ light chain genes obtained from a cDNA library of a rheumatoid arthritis (RA) synovial tissue. A salient feature of our results is the high frequency utilization of germline VκI family genes, especially the O2/O12 gene (38%). All κ V‐regions showed extensive somatic hypermutation with 5.4% of an average mutation rate. Replacement to silent mutation (R/S) ratio in the complementarity determining region (CDR) was > 2.9 in 12 out of 16 clones, indicating that the majority of the RA synovial B cells had undergone affinity maturation. However, the four other clones showed R/S ratios of < 2.9 in the CDR despite a high mutation rate. In contrast to the previous reports, long CDR3 was not a characteristic feature of these clones. In summary, these data show the high frequency utilization of the germline O2/O12 gene and a high rate of mutation with an evidence of antigen selection in most of the Ig κ genes expressed in the RA synovium.
Non‐Lethal Viral Challenge of Influenza Haemagglutinin and Nucleoprotein DNA Vaccinated Mice Results in Reduced Viral Replication
Authors: Cox R. J. , Mykkeltvedt E. , Robertson J. , Haaheim L. R.
Published: March 2003

Influenza DNA vaccines have been widely studied in experimental animal models and protection documented after lethal viral challenge. In this study, we have investigated the humoral response after a non‐lethal viral challenge of mice vaccinated with plasmids encoding the influenza haemagglutinin (HA) or nucleoprotein (NP) genes. BALB/c mice were immunized intramuscularly with three doses (100 µg) of HA, NP or backbone plasmid at 3‐week intervals, or alternatively infected intranasally, before being challenged with homologous virus 13 weeks later. Mice were then sacrificed at weekly intervals and the antibody‐secreting cell response was examined systemically (spleen and bone marrow) and in the respiratory tract (nasal associated lymphoid tissue (NALT) and lungs). Sera were collected after each dose of vaccine and at sacrifice and analyzed by ELISA, haemagglutination inhibition and virus neutralization assays. We found that previous viral infection apparently elicits sterilizing immunity. Vaccination with HA or NP DNA significantly reduced viral replication in the nasal cavity after viral challenge, however, increases in serum antibody titres were observed after challenge. Prior to challenge, specific antibody‐secreting cells were observed in the systemic compartment after HA or NP DNA vaccination but were also found in the NALT after viral challenge. In conclusion, intramuscular DNA vaccination resulted in immunological memory in the systemic compartment, which was rapidly reactivated upon viral challenge.
Human CD8+ CTL Recognition and In vitro Lysis of Herpes Simplex Virus‐Infected Cells by a Non‐MHC Restricted Mechanism
Authors: Garland R. J. , El‐Shanti N. , West S. E. , Hancock J. P. , Goulden N. J. , Steward C. G. , Rowbottom A. W.
Published: March 2003

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA‐DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV‐infected lymphoblastoid cell line (LCL) targets via a non‐major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium‐dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic‐infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non‐MHC restricted lysis‐associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcγR‐bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non‐MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.
Fc Receptors are Critical for Autoimmune Inflammatory Damage to the Central Nervous System in Experimental Autoimmune Encephalomyelitis
Authors: Abdul‐Majid K‐B. , Stefferl A. , Bourquin C. , Lassmann H. , Linington C. , Olsson T. , Kleinau S. , Harris R. A.
Published: March 2003

Multiple sclerosis (MS) is simulated by various forms of experimental autoimmune encephalomyelitis, in which T cells, antibodies, cytokines and complementary factors interact with the central nervous system (CNS) myelin proteins and lead to inflammatory damage. We investigated the role of Fc receptors (FcRs), which link the cellular and humoral branches of the immune system, in myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE), using two different FcRγ knockout DBA/1 mice. The first knockout were the FcRγ chain‐deficient mice, which lack FcγRI, FcγRIII and FcεRI, while the second knockout mice lack only FcγRII. The lack of FcγRII enhanced the disease susceptibility with associated increased CNS demyelination. While FcRγ+/+ DBA/1 mice also developed pronounced CNS infiltration and myelin destruction, FcRγ−/− littermates were protected despite initial peripheral autoimmune responses to MOG. In vitro analyses revealed equivalent potentials of fluid phase phagocytosis of myelin and MOG in bone‐marrow macrophages derived from both FcRγ+/+ and FcRγ−/− mice, while MOG‐immunoglobulin (Ig)G immune complexes were only internalized by FcRγ+/+ macrophages. This was associated with cellular activation in FcRγ+/+ but not FcRγ−/− macrophages, as assessed by the activation of intracellular mitogen activated protein (MAP)‐kinase signalling elements. We propose that protection from EAE in FcRγ‐deficient mice is due to the inefficient antigen processing/presentation of myelin proteins during the induction of secondary immune responses locally in the CNS, which leads to demyelination. This demonstrates the importance of FcR in the promotion of autoimmune inflammation of the CNS and highlights the therapeutic possibility of treatment of MS with FcR‐directed modalities.
Diminished Th1 and Th2 Cytokine Production in Healthy Adult Nonresponders to Recombinant Hepatitis B Vaccine
Authors: Kardar G. A. , Jeddi‐Tehrani M. , Shokri F.
Published: March 2003

Vaccination of healthy adults with recombinant hepatitis B (rHB) vaccine fails to induce a protective antibody response in a proportion of individuals. Imbalanced T‐helper (Th)1/Th2 response has been attributed to the lack of specific antibody response to rHB vaccine. In this study, in vitro production of interleukin (IL)‐2, interferon (IFN)‐γ and IL‐10 was investigated in Iranian healthy adults vaccinated with rHB vaccine. Peripheral blood mononuclear cells (PBMC) were isolated from 18 high responders and eight nonresponders and stimulated with rHB antigen or phytohaemaglutinin (PHA) mitogen. The cytokines were quantitated in culture supernatants by sandwich enzyme‐linked immunosorbent assay (ELISA). Our results demonstrated a significant decrease in the production of IL‐2, IFN‐γ and IL‐10 (P < 0.005) in response to rHB antigen. The levels of all cytokines induced by PHA were similarly represented in both groups of vaccinees. These findings suggest that unresponsiveness to rHB vaccine may be owing to inadequate Th1‐ and Th2‐like cytokine production.
Polymorphisms in Interleukin‐1β and Interleukin‐1 Receptor Antagonist Genes and Malaria in Ghanaian Children
Authors: Gyan B. , Goka B. , Cvetkovic J. T. , Perlmann H. , Lefvert A.‐K. , Akanmori B. , Troye‐Blomberg M.
Published: March 2003

We have investigated the possible associations between polymorphisms in two interleukin‐1 (IL‐1) genes and severity of Plasmodium falciparum malaria in Ghanaian children with cerebral malaria, severe anaemia or uncomplicated malaria and controls. There was no significant difference in genotype and allele frequencies in IL‐1β exon 5 or interleukin‐1 receptor antagonist (IL‐1ra) polymorphisms between the studied groups, suggesting that the two polymorphisms may not be involved in the pathogenesis of severe malaria. When parasitaemias in uncomplicated malaria patients were evaluated, a significantly higher level of parasitaemia was observed among carriers of IL‐1β A2 allele as compared with noncarriers of this allele (P = 0.01). The mean parasitaemia in an age‐matched asymptomatic group did not reveal such associations. These data suggest that IL‐1β exon 5 allele 2 may play a possible role in the clinical outcome of uncomplicated malaria.