bluuebunny/crossref_metadata_2025 huggingface dataset:
Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein
Authors: Escolar Lucía , Lorenzo Víctor de , Pérez‐Martíín José
Published: November 2003

The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+‐responsive Fur (ferric uptake regulation) protein has been investigated. In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences. However, binding to the primary target site suffices for the entire repression effect. Comparison of transcription patterns generated with run‐off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal −35/−10 hexamers of the promoter region was fully prevented by Fur‐Mn2+ bound to its primary site. Similarly, promoter‐bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur‐Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription. The high affinities of either protein for the promoter produce, in practice, a first‐come, first‐served effect that helps the system to respond instantly to changes in the iron status of the cells.
Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli
Authors: Mourez M. , Skouloubris S. , Betton J.‐M. , Dassa E.
Published: November 2003

We analysed the effects of the overproduction of parts or all of a multisubunit ATP‐binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, σ24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.
Two‐component regulatory system involved in transcriptional control of heavy‐metal homoeostasis in Alcaligenes eutrophus
Authors: van der Lelie Daniel , Schwuchow Tanja , Schwidetzky Uta , Wuertz Stefan , Baeyens Wilfried , Mergeay Max , Nies Dietrich H.
Published: November 2003

The czc determinant, which mediates resistance to Co2+, Zn2+ and Cd2+ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two‐component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described). Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR). Transcription of czcCBA and of the URR was regulated by heavy‐metal cations. DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR. Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion. Expression of both genes is also regulated by heavy metals. The genes of the URR yielded three mRNAs of approx. 1200, 500 and 200 nucleotides, respectively. The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.
Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence
Authors: Chesnokova Olga , Coutinho John B. , Khan Imran H. , Mikhail Maurice S. , Kado Clarence I.
Published: November 2003

Agrobacterium tumefaciens produces flagella that are arranged circumthecally near one end of the bacilliform cell. The flagella are required for motility to facilitate reaching the root surface, and possibly aid in orientating the bacterial cells at various sites for infection. We have identified three flagella genes designated flaA, flaB, and flaC. Mutations in flaA, flaB and flaC result in abberant swimming behaviour. Electron microscopic examination of these mutants revealed the defective flagella. A non‐motile, bald mutant strain was generated by deleting all three fla genes. Nucleotide sequencing of flaA, flaB, and flaC showed that they have a potential coding capacity for polypeptides of 307, 321, and 314 amino acid residues, respectively. The predicted amino acid sequences of the A. tumefaciens FlaA and FlaB proteins are similar (66% average identity) to the FlaA and FlaB proteins encoded by flaA and flaB genes, respectively, in Rhizobium meliloti. There was no counterpart FlaC protein reported in R. meliloti, but the A. tumefaciens FlaC is similar in amino acid sequence to the R. meliloti FlaA (59.8% identity) and FlaB (66.7% identity). Distinct from FlaA and FlaB of R. meliloti is the absence of histidine and cysteine residues and their shorter length (by 88 amino acid residues fewer than FlaA and FlaB of R. meliloti ). The transcriptional start sites of each fla gene determined by primer extension revealed consensus‐sequence boxes representing potential binding sites for σ28 RNA polymerase (RNAP) upstream of the transcriptional start of each fla gene. Besides the potential σ28‐binding site upstream of flaC, also present are additional putative conserved sequences, GC at −11 and GG at −21 from the transcriptional start, that resemble potential binding motifs for σ54. Because the σ54 promoter is associated with genes regulated by physiological changes in various bacteria, the flaC gene might be similarly regulated in response to A. tumefaciens responding to host plant stimuli. Virulence studies showed that the bald strain was consistently reduced in virulence below that of the parental wild‐type strain by at least 38%. The difference is statistically significant and suggests that the flagella may play a role in facilitating virulence.
Superior laryngeal nerve stimulation in the cat: effect on oropharyngeal swallowing, oesophageal motility and lower oesophageal sphincter activity
Authors: Beyak M. J. , Collman P. I. , Valdez D. T. , Xue S. , Diamant N. E.
Published: November 2003

Superior laryngeal nerve (SLN) stimulation can activate the brainstem swallowing mechanism to produce a complete swallowing sequence consisting of oropharyngeal, oesophageal and lower oesophageal sphincter (LOS) components. However, little is known of the effect of SLN stimulation (peripheral‐sensory input from the pharynx) on the characteristics of oesophageal motor activity, especially in the smooth muscle portion. The present study examined the effect of varying stimulus train length and frequency on each of the three components of the reflex. Acute studies were performed in urethane anaesthetized cats. Oesophageal motility was monitored using conventional manometric techniques, and oropharyngeal swallowing by the mylohyoid electromyogram. SLN stimulus train length (1–10 sec) and frequency (5–30 Hz) were varied independently. Increased train length or frequency resulted in (1) an increase in oropharyngeal swallowing and incidence of the complete swallowing response, (2) an increase in latency to onset of the oesophageal peristaltic wave, (3) reduction of the amplitude of the evoked peristaltic contraction in the smooth muscle portion, without altering its velocity, (4) increased LOS relaxation, and increased LOS after‐contraction. The LOS contraction was abolished by atropine (100 μg kg−1). Therefore, increased SLN stimulation not only results in excitation of the central swallowing program and the oropharyngeal stage of swallowing, but has major effects on the oesophageal and LOS stages of swallowing. Afferent SLN stimuli can impact on the control mechanisms for each stage, to inhibit or excite the stages in different ways.
Colonic migrating motor complexes (CMMCs) in the isolated mouse colon
Authors: Fida R. , Lyster D. J. K. , Bywater R. A. R. , Taylor G. S.
Published: November 2003

Spontaneous contractions were recorded from the circular muscle layer at three sites along the isolated mouse colon. The interval between contractions was ≈ 4.5 min. The mean duration of the contractions ranged from 26 sec in the distal colon to 45 sec in the proximal colon. Contractions migrating more than half the length of the colon were termed colonic migrating motor complexes (CMMCs). Over 90% of tissues demonstrated migration predominantly in an aboral direction. Hyoscine (10−6 m) decreased the amplitude of the CMMCs by at least 40% but had no significant effect on the interval or duration of the CMMCs. Nifedipine (10−6 m) significantly decreased the amplitude of the CMMCs by 95% but did not alter the duration or the interval between the CMMCs. Hexamethonium (5 × 10−4 m) and tetrodotoxin (TTX; 2 × 10−6 m) abolished all CMMC activity. TTX increased the resting tone of the preparations. Nitro‐L‐arginine (10−4 m) increased the resting tone of the preparations and significantly decreased the interval between the CMMCs by ≈ 80% but had no significant effect on the duration of the CMMCs. The results suggest CMMCs migrate predominantly in an aboral direction and are neurogenic in origin. Nitric oxide may be involved in maintaining inhibition of the muscle between CMMCs.
5‐HT activates neural reflexes regulating secretion in the guinea‐pig colon
Authors: Cooke H. J. , Sidhu M. , Wang Y.‐Z.
Published: November 2003

The role of 5‐hydroxytryptamine (5‐HT) in neural reflexes regulating secretion was examined in muscle‐stripped segments of guinea‐pig colon set up in modified flux chambers. A 15‐μL pulse of 5‐HT (100 μM) to the mucosal bath (1.5 mL), which was continuously perfused, evoked an increase in short‐circuit current (Isc). The 5‐HT‐induced increase in Isc was inhibited by tetrodotoxin, N‐acetyl‐5‐hydroxytryptophyl‐5‐hydroxytryptophan amide (5‐HTP‐DP), GR82334 and atropine, but not by tropisetron. 5‐HTP‐DP reduced the response to a 5‐HT pulse over the concentration range of 1 nM to 1 μM. The Isc response to a 5‐HT pulse was unaffected by the cyclooxygenase inhibitor, piroxicam. This contrasted with a reduction in the Isc response to mucosal stroking with a brush by piroxicam. The results suggest that a 5‐HT pulse, like mucosal stroking, activates a secretory reflex that includes tachykinin and cholinergic neurons but, unlike mucosal stroking, does not release prostaglandins.
Gastric contractions, secretions and injury in cold restraint
Authors: Ephgrave K. S. , Cullen J. J. , Broadhurst K. , Kleiman‐Wexler R. , Shirazi S. S. , Schulze‐Delrieu K.
Published: November 2003

The clinical syndrome of stress ulceration has been studied for years using rodent cold restraint stress models, although the pathogenesis of the characteristic focal gastric mucosal lesions produced in these models has been controversial. We used gastric strain gauges to characterize fully the gastric motility effects of a 4‐h cold restraint protocol, and we determined the relationship of variations in gastric contents and in gastric contractions to the amount of gastric mucosal injury. Additionally, we examined rat stomachs histologically, and determined the location of focal haemorrhagic mucosal lesions on the mucosal rugae. We found a consistent relationship between force of gastric contractions and gastric mucosal injury, and also a relationship between the initial duration of contractions during restraint and ultimate mucosal injury. Volume, acidity and mucus in the gastric contents were unrelated to mucosal injury. The majority (91%) of the mucosal lesions had some relationship to a rugal fold, with 59% of all lesions at the base of a rugal fold. Thus, the mechanical forces of gastric hypercontractility may contribute to the gastric mucosal injury of rodent cold restraint models.
Social change and conservation misrepresentation in Africa
Authors: Spinage Clive
Published: November 2003

Concomitant with the increasing denouncement of African game legislation as inappropriate law imposed by a former colonial authority, is the attack upon traditional, i.e. total protection, practice of conservation. It is increasingly argued by a school of neo-populist thinkers, that local people should be allowed to exploit protected areas in accordance with their own traditions and beliefs. Examples of alleged injustice or practice are consistently misrepresented with a view to replacing traditional conservation practice with left-wing political dogma, proponents claiming a mandate from the Caracas 1992 IVth World Congress on National Parks and Protected Areas.
The black lion tamarinLeontopithecus chrysopygus– its conservation and management
Authors: Mamede-Costa Ana Carolina , Gobbi Nivar
Published: November 2003

The black lion tamarinLeontopithecus chrysopygusoriginally occurred throughout a large part of the Atlantic forest in the west of the state of São Paulo, Brazil. Today, however, it is restricted to a few isolated forest fragments as a result of deforestation caused by cattle ranching, and urban and agricultural expansion, especially in this century. One of its last strongholds is a small gallery forest at Lençóis Paulista in the west-central part of the state. The authors report on a long-term study of this small and isolated population, aimed particularly at providing a basis for the intensive managementand conservation of the species and its habitat.
Comparison of complement activation in vitro by different Echinococcus granulosus extracts
Authors: IRIGOÍN FLORENCIA , WÜRZNER REINHARD , SIM ROBERT B. , FERREIRA ANA M.
Published: November 2003

In the present study we have investigated and compared in vitro the specific complement (C) activating activity of three metacestode preparations of Echinococcus granulosus. Extracts from hydatid cyst fluid (HCF‐ext), protoscoleces (PSC‐ext) and hydatid cyst membrane (HCM‐ext) activated human C producing C3 conversion and generating the C5b6 complex and the terminal C complex (TCC). HCM‐ext showed much lower C activating activity than PSC‐ext and HCF‐ext. Moreover, its ability to generate C5b6 and TCC was lower than its ability to convert C3. On the other hand, PSC‐ext and HCF‐ext proved to be good C activators when their specific C activating activities were compared with that of inulin. However, PSC‐ext produced lower levels of TCC than those produced by HCF‐ext, in spite of the fact that both produced practically the same levels of C3d and C5b6. These results may be consistent with the existence of several mechanisms of C modulation involved in the defence of the parasite against host C damage.
Antigenic polypeptides of Echinococcus granulosus oncospheres and definition of protective molecules
Authors: HEATH DAVID D. , LAWRENCE STEPHEN B.
Published: November 2003

Immunoblotting and in vitro oncosphere‐killing were used to identify a putative protective molecule in Echinococcus granulosus mature oncospheres. A range of sera from sheep that had been shown to be protected against E. granulosus, and from those that were not, were tested. The sera used were obtained from sheep hyperimmunized with E. granulosus oncospheres, or immunized with oncosphere non‐denatured extract, with immature oncosphere extract or with denatured extracts of oncospheres. Results indicated the involvement of native antigens of 23, 25, 30, 34 and 40 kDa in the protective response to E. granulosus infection. The rapid appearance of antibodies to the 23, 25 kDa antigens, their association with early onset of protection and in vitro oncosphere lysis by affinity‐purified antibodies obtained from these fractions, indicated that these antigens contained protective epitopes. Final confirmation was provided by immunization of sheep with fractions prepared by preparative SDS/PAGE, and challenge infection. Only the fraction containing the 23 and 25 kDa molecules was able to stimulate protection. Antisera against this pair of molecules should provide a useful probe for screening an E. granulosus oncosphere cDNA library to identify clones expressing protective molecules.
Kinetics and mechanism of effector focus formation in the lungs of mice vaccinated with irradiated cercariae of Schistosoma mansoni
Authors: SMYTHIES LESLEY E. , BETTS CATHERINE , COULSON PATRICIA S. , DOWLING MARY‐ANN , WILSON R. ALAN
Published: November 2003

The sequence of events involved in effector focus formation around challenge schistosomula in the lungs of mice vaccinated with radiation‐attenuated cercariae of Schistosoma mansoni has been characterized following intravenous administration of lung stage larvae. Histopathological analysis of the lungs of vaccinated animals revealed that infiltrating cells were present around larvae within 24 h. The main increment in cell recruitment occurred between 2 and 4 days, with foci reaching maximal diameter on day 8. No additional infiltration of the airways was detected by bronchoalveolar sampling before day 4 when the maximum number of cells, predominantly lymphocytes, was recovered. In contrast, responses in challenge control animals were relatively slight prior to day 12. IFNγ was the major cytokine in airway cultures from vaccinated mice, the greatest increment in production coinciding with peak cell recruitment. A similar pattern of IFNγ mRNA expression was observed in whole lung extracts, highlighting the dominance of Th1 responses in the effector mechanism. The slow start to focus formation may be due to the need for antigen, released by the intravascular parasite, to be translocated across the endothelium, processed by accessory cells and presented to the helper T cells which orchestrate the effector mechanism. The delay is of the same order as the period of development which the parasite must undergo in the lung, to facilitate further migration. This similarity in the timing may explain why some larvae are able to avoid the consequences of the pulmonary effector response.
Human isotype antibody responses to an Onchocerca volvulus glutathione S‐transferase
Authors: Salinas G. , Sinha K. , Cooper J.P. , Whitworth J.A.G. , Taylor D.W.
Published: November 2003

Human isotype specific antibody responses to a recombinant π‐class glutathione S‐transferase (Ov24) from Onchocerca volvulus were assesed by ELISA, using a large and well‐characterized bank sera (n=238) from an hyper‐endemic area of moderate intensity from Sierra Leone. IgG1, IgG4 and IgA responses, but neither IgG2 nor IgE response, to Ov24 were detected in infected subjects. The relationships between Ov24 antibody levels and skin microfilarial density, number of nodules, age, sex, eosinophil counts and clinical sign of reactive and chronic pathology were analysed using Pearson’s correlation coefficient. Significant correlations between both IgA and IgG3 antibody levels and age were found (P<0.01). Although no firm conclusions could be drawn from this study sample regarding the relationships between antibody levels and parasite load or clinical status, a negative correlation (P=0.06) between Ov24 IgG3 antibody levels and microfilarodermia was found.
Protection of mice against Schistosoma mansoni infection by passive transfer of sera from infected rabbits
Authors: FALLON P.G. , FOOKES R.E. , DOENHOFF M.J.
Published: November 2003

Sera from rabbits infected with unattenuated Schistosoma mansoni cercariae conferred significant levels of protection against S. mansoni challenge (P < 0.001) after passive transfer to mice. Infected rabbit sera were only effective in conferring protection when transferred during the first week of infection, and were not effective when administered against liver‐stage worms. Immunoglobulins isolated from the infected rabbit sera with Protein A‐Sepharose were shown to be responsible for the transfer of protection to mice. Immunofluorescence studies demonstrated that the sera were more reactive against the surface of three hour‐old mechanically transformed schistosomula than against the surfaces of lung‐stage schistosomula. The sera from infected rabbits reacted polyspecifically against antigens in cercaria, schistosomula, and the worm and egg stages of the S. mansoni life‐cycle. The host parasite relationship of S. mansoni in the rabbit is discussed.
Immunological responses to antigen B from Echinococcus granulosus cyst fluid in hydatid patients
Authors: IOPPOLO S. , NOTARGIACOMO S. , PROFUMO E. , FRANCHI C. , ORTONA E. , RIGANO R. , SIRACUSANO A.
Published: November 2003

The immunological reactivity of Echinococcus granulosus antigen B was evaluated in 30 hydatid patients. Antigen B was purified from sheep hydatid cyst fluid by electroelution from a non‐reducing SDS‐PAGE gel (AgB). In ELISA and immunoblotting (IB), determining antibody production in sera from patients with hydatid disease and with other parasitic infections, purified AgB showed higher specificity than a partially purified antigen named pH5PPT (100% vs 83% in pH5PPT‐ELISA and 58% in pH5PPT‐IB). AgB‐IB achieved higher sensitivity than AgB‐ELISA (80% vs 63%). All AgB‐IB positive sera recognized the 12 kDa subunit. Qualitative AgB‐IB assessment of IgG isotype responses identified IgG4 as the predominant isotype (87%). The other isotypes showed a lower percentage of positive reactions: IgG1, 33%; IgG2, 21%; and IgG3, 17%. PBMC proliferative assay revealed a cellular response to AgB in 100% of patients’ PBMC. These findings confirm antigen B, especially its smallest subunit, as a good diagnostic molecule.
Immunopathology of the uveitis in canine leishmaniasis
Authors: GARCÍA‐ALONSO M. , BLANCO A. , REINA D. , SERRANO F.J. , ALONSO C. , NIETO C.G.
Published: November 2003

Particular immunopathological features and their effects on the vascular permeability of different ocular structures were analysed in two dogs naturally infected by Leishmania infantum. The existence of specific anti‐Leishmania immunoglobulin G (IgG) in the aqueous humour was confirmed by the ELISA technique. There was no correlation between antibody levels in the aqueous humour and the related serum. The histopathological study of the eyes showed the existence of lesions in various ocular structures. The ciliary processes, ciliary body, sclerocorneal limbus, iris and lacrimal duct showed intense inflammatory zones with lymphocyte infiltrates, plasmatic cells and macrophages with amastigote forms of Leishmania. In addition vasculitis with dilation and thrombi were also detected in both cases, with consequent oedema and hyalinization. The immunohistochemistry analysis revealed the presence of granular and diffuse IgG deposits in the ciliary body, ciliary processes, sclerocorneal limbus and iris. Furthermore, numerous thrombosed vessels were observed in the sclerocorneal zone and iris. Complement 3 (C3) fraction deposits were not present in the ocular structures. The present data suggest that the ocular lesions may have an immunopathological origin.
Evidence that cellular immune responses to soluble and membrane associated antigens are independently regulated during human schistosomiasis mansoni
Authors: BAHIA‐OLIVEIRA L.M.G. , SIMPSON A.J.G. , ALVES‐OLIVEIRA L.F. , CARVALHO‐QUEIROZ C. , SILVEIRA A.M.S. , VIANA I.R.C. , CUNHA‐MELO J.R. , HAGAN P. , GAZZINELLI G. , CORREA‐OLIVEIRA R.
Published: November 2003

We have made a comparative analysis of human cellular and antibody responses to membrane associated adult worm antigens (Mb‐A), soluble adult worm antigens (SWAP) and soluble egg antigens (SEA) derived from Schistosoma mansoni. Chronically infected patients with the intestinal (I) and hepatosplenic (HS) forms of the disease as well as non‐infected putative immune ‘endemic normals’ (EN), were studied. We observed that the cellular responses, of individuals, to the two adult worm preparations, SWAP and Mb‐A, may be distinct and can be related to the occurrence of resistance or pathology. The resistant group (EN) presented higher levels of both cellular proliferation, and IFN‐γ production, in response to Mb‐A as compared with SWAP whereas HS individuals presented higher levels of cellular proliferation to SWAP as compared with Mb‐A. Individuals with intestinal disease had similar levels of proliferation to both antigens. The response to SEA by all groups was generally similar, and not predictive of any clinical form. The specific antibody response to the three antigens were in general higher among infected patients than in resistant EN individuals. These results support the hypothesis that the response to adult worm antigens may be pivotal in determining both the development of resistance and severity of disease.
In vitro stimulation of naive mouse lymphocytes by Heligmosomoides polygyrus adult worm antigens induces the production of IgG1
Authors: ROBINSON MICHAEL , GUSTAD THOMAS R.
Published: November 2003

The production of high levels of IgG1, by mice chronically infected with the parasitic nematode Heligmosomoides polygyrus, has been documented for a number of years. In order to investigate this phenomenon, naive lymphocytes from B10.D2 mice were incubated in vitro with H. polygyrus adult worm homogenate (AWH) and the culture supernatants examined for immunoglobulin production. Stimulation of pooled naive splenocytes by AWH was found to produce IgG1, but not IgM, in an antigen dose dependent manner. Identical stimulation of splenocytes of individual inbred mice, indicated that this effect was reproducible but with considerable variation between mice. When the IgG1 produced was tested for specificity, it was found that there was little evidence that the immunoglobulin produced was able to bind to the inducing parasite antigens. Analysis of purified T cells reconstituted with splenocytes, demonstrated that T cells were the target lymphocytes of the stimulating molecule, contained within AWH. These results show that H. polygyrus AWH can induce the production of non‐parasite specific IgG1 from naive splenocytes and that this production is crucially dependent upon the cell content of the in vitro culture. Furthermore, the production of IgG1 is not proportional to the degree of lymphocyte proliferation. It is suggested that at least part of the hypergammaglobulinaemia produced during a primary H. polygyrus infection, is due to this non‐specific stimulation of mouse lymphocytes by the parasite.
Can software be used to predict antigenic regions in Plasmodium falciparum peptides?
Authors: DE FÁTIMA FERREIRA‐DA‐CRUZ MARIA , GIOVANNI‐DE‐SIMONE SALVATORE , BANIC DALMA MARIA , CANTO‐CAVALHEIRO MARILENE , CAMUS DANIEL , TADEU DANIEL‐RIBEIRO CLAUDIO
Published: November 2003

We compared the antigenicity of p126 Plasmodium falciparum peptides with predicted antigenic regions identified using the methods described by Garnier et al. (1978) and Chou & Fasman (1974). For this purpose nine different P. falciparum peptides were synthesized in accordance with the deduced amino acid sequence of the p126 gene, and their reactivity was tested using an enzyme linked immunosorbent assay against sera from individuals with a natural malaria infection. Both predictive methods gave similar antigenic‐index scores, however, a comparison of these predictive results with data obtained by ELISA showed that the probability of a correct prediction was only around 45% for both cases. Thus, in our view computer softwares could not be used in isolation for screening purposes, and other parameters must also be taken into account when using such software to assess antigenicity.
The role of anti‐variable surface glycoprotein antibody responses in bovine trypanotolerance
Authors: WILLIAMS D.J.L. , TAYLOR K. , NEWSON J. , GICHUKI B. , NAESSENS J.
Published: November 2003

It has been reported that some breeds of cattle such as the N'Dama mount a more effective antibody response to the variable surface glycoprotein coat of trypanosomes and that this may contribute to their ability to control the infection. Thus we have investigated antibody responses to surface exposed epitopes of the variable surface glycoprotein in Trypanosoma congolense‐infected N'Dama (trypanotolerant) and Boran (susceptible) cattle. Similar titres and isotypes were found in both N'Damas and Borans indicating that trypanotolerance is not associated with superior antibody‐mediated destruction of trypanosomes. However, significant differences in antibody responses to cryptic VSG epitopes and non‐trypanosome antigens were identified. Trypanosusceptible Boran cattle had low IgG1 responses to cryptic epitopes but high IgM responses to non‐trypanosome antigens such as β‐galactosidase. In contrast the N'Dama cattle had significantly higher IgG1 responses to cryptic VSG epitopes and negligible responses to β‐galactosidase. These results indicate differences in the induction of anti‐trypanosome immune responses between trypanotolerant and susceptible cattle infected with T. congolense
Analysis of the immune response elicited by a Multiple Antigen Peptide (MAP) composed of two distinct protective antigens derived from the parasite Schistosoma mansoni
Authors: FERRU I. , GEORGES B. , BOSSUS M. , ESTAQUIER J. , DELACRE M. , HARN D.A. , TARTAR A. , CAPRON A. , GRASSMASSE H. , AURIAULT C.
Published: November 2003

In this report we analyse the immune response elicited by a Multiple Antigen Peptide (MAP), containing three peptide sequences derived from two distinct vaccine candidates against schistosomiasis; the Schistosoma mansoni 28 kDa Gluthatione‐S‐Transferase (Sm28GST) and the Schistosoma mansoni Triose‐Phosphate‐Isomerase (sTPI). We examined the immunogenicity of this construct, named MAP ‘DA’, in three distinct mouse strains. The B‐cell response, studied by measuring the production of different IgG isotypes, was mainly directed against the peptide derived from the Sm28GST, but also against the whole Sm28GST protein. In contrast, the T‐cell response, as assessed by proliferation assay and cytokine mRNA expression, was directed against the MAP construct, the peptides derived from the sTPI protein and the whole sTPI protein. Significantly, T‐cells from all MAP ‘DA’‐immunized mice, restimulated in vitro was the sTPI antigen, expressed IFN‐γ specific messengers. This cytokine has been described to play a major role in the reduction of the Schistosoma mansoni pathology. We thus demonstrate that a single MAP construct, composed of peptides from distinct antigens of Schistosoma mansoni, induced a B‐ and T‐cell response, including production of potentially protective IFN‐γ, irrespective of the MHC background.
Salivary antigens of the cat flea, Ctenocephalides felis felis
Authors: LEE S.E. , JACKSON L.A. , OPDEBEECK J.P.
Published: November 2003

The cat flea, Ctenocephalides felis felis, is the major cause of flea bite hypersensitivity (FBH) in dogs and cats, yet little progress has been reported on identifying the antigens responsible. We obtained flea salivary antigens by washing secretions from containers probed by the mouthparts of fleas, and by extracting whole flea salivary glands. Mice were exposed to feeding fleas to generate antibodies to salivary antigens injected in vivo. The sera were tested for antibodies against the salivary antigens described and against a whole flea extract; in indirect ELISA, antibodies to salivary secretions were detected in 60% of the sera from mice exposed to feeding fleas. These sera identified four protein bands at apparent MW 56, 54, 42 and 40 K which corresponded to prominent protein bands in whole salivary gland extracts identified by protein staining after SDS‐PAGE. Fixed sections of whole fleas exposed to the antisera showed that only structures within the salivary glands were identified. The salivary secretions and gland extracts are now being used to study immune responses of dogs suffering from FBH.
Serological reactivity to heat shock protein 70 in patients with hydatid disease
Authors: COLEBROOK AMANDA L. , .LIGHTOWLERS MARSHALL W
Published: November 2003

Heat shock protein 70 (hsp70) was chosen as a model antigen with which to investigate autoantibody production in humans with cystic hydatid disease. Levels of specific serum antibody were assessed in the sera of patients with surgically confirmed infection with E. granulosus and sera from non‐infected controls. Antigens used were human hsp70, obtained from K562 cells grown in culture, and E. granulosus hsp70 obtained by expression of the full length protein in Escherichia coli following cloning of the associated mRNA. Antibody reactivity to human hsp70 was detected in the sera of only a small proportion of hydatid patients (10%) as well as a similar proportion of sera from age matched controls. Specific antibodies reactive with E. granulosus hsp70 were detected in 60% of hydatid patients, although some samples (21%) from healthy controls also reacted with E. granulosus hsp70, the level of reactivity was significantly higher in hydatid patients. This report identifies E. granulosus hsp70 as an immunogen during human hydatid infection but, despite its having a predicted 81% protein sequence homology with human hsp70, it does not appear to induce autoimmune reactivity against the homologous human protein.
Non‐specific binding of IgG1 to Heligmosomoides polygyrus: adult worm homogenate superantigen is a target for immunoglobulin‐induced inhibition
Authors: ROBINSON MICHAEL , GUSTAD THOMAS R. , ERICKSON MICHELE R. , FERGUSON JENNIFER L. , DAVID CHELLA S.
Published: November 2003

Previous evidence had indicated that selected antigens contained in H. polygyrus adult worm homogenate (AWH) could bind non‐specifically to mouse IgG1. To determine whether H. polygyrus superantigen was one of these binding molecules, an inhibition assay was carried out using monoclonal antibodies (MoAb) to block the in vitro superantigen response. The results indicated that non‐specific IgG1 binding could inhibit the cellular response to the superantigen. This assumption was tested using affinity chromatography to extract those antigens which bound non‐specifically to mouse IgG1. Both the protein fraction which bound to the column and the unfractionated AWH demonstrated superantigen activity, as described previously. In contrast, the unbound fraction contained no superantigen activity. None of the tested fractions exhibited non‐specific mitogen activity. These results indicate that the superantigen produced by H. polygyrus binds to host IgG1 of any specificity and this binding can inhibit further host recognition of this molecule. Additionally, it was demonstrated that an apparently similar superantigen is also contained in L4 homogenate, and is strongly represented in the excretory/secretory (E/S) proteins produced by both adult and L4 parasitic stages. Therefore, it is probable that H. polygyrus superantigen influences the host during both the L4 and adult stages of the life‐cycle.
Antibody isotype responses to Trichuris infection in humanized SCID mice
Authors: ELSE KATHRYN J. , BETTS CATHERINE J.
Published: November 2003

We have developed a humanized SCID mouse model of trichuriasis involving the injection of peripheral blood lymphocytes (PBLs) from normal healthy donors into C. B.‐17 severe combined immunodeficiency (SCID) mice followed by vaccination and infection of these mice with the murine species of Trichuris, Trichuris muris. Optimal results with respect to parasite specific antibody production and peripheral engraftment were achieved by injecting intraperitoneally 2 × 107 PBLs which had been incubated overnight on anti‐human CD3 coated plates. Mice were immunized three weeks post reconstitution with parasite antigen in Freund's Incomplete Adjuvant and infected two weeks later. At autopsy human T cells could be detected in the spleens of engrafted animals and anti‐T. muris antibody detected. The dominant IgG isotype responses were shown to be IgG1 and IgG2, providing a similar IgG isotype profile to that seen in humans infected with T. trichiura in the field. In several cases engrafted animals showed the remarkable ability to expel their parasite load. The model will thus be useful for analysing human immune responses to trichuriasis under highly controlled laboratory conditions impossible to achieve in the field.
Elimination of a primary schistosome infection from rats coincides with elevated IgE titres and mast cell degranulation
Authors: CUTTS L. , WILSON R.A.
Published: November 2003

Schistosomes are eliminated from laboratory rats around 28 days post‐infection, whilst they are still resident within the hepatic portal distributaries of the liver. We have previously shown that their presence in this location is accompanied by an intense mastocytosis. We have investigated the potential relationships between IgE responses, the allergenicity of schistosome antigens, mast cell responsiveness, and worm elimination. Total and specific IgE were measured using an ELISA and a functional assay based on 3H serotonin release from activated rat basophilic leukemia cells (RBL‐SRA), respectively. Both assays revealed that infected rats produced elevated IgE titres relative to naive animals. At days 28 and 35, mixed‐sex infections stimulated a higher total IgE than male‐only infections. IgE was affinity purified from rat infection serum and used to probe a fractionated soluble worm antigen preparation (SWAP) by Western blotting. Two allergenic products were detected of M
r
67 and 36–38 kDa, the former having the same molecular weight as a previously identified secretory protein. IgE from mixed‐sex schistosome infections bound strongly to the 36–38 kDa molecule, compared to the relatively weak binding exhibited by IgE from male‐only infection serum. Since eggs were not recovered from the infected rats, this reactivity was attributed to the greater release of allergens from female worms. Results from the RBL‐SRA showed that female SWAP was a more effective trigger of mast cell degranulation in vitro, for equal amounts of protein. This enhanced allergenicity was ascribed to the relative abundance of carbohydrate moieties. Our results support a role for IgE, and mast cell degranulation in the elimination of a primary schistosome burden from rats.
Elevated IL‐10 mRNA expression and downregulation of Th1‐type cytokines in microfilaraemic individuals with Wuchereria bancrofti infection
Authors: RAVICHANDRAN MANICKAM , MAHANTY SIDDHARTHA , KUMARASWAMI V. , NUTMAN THOMAS B. , JAYARAMAN KUNTHALA
Published: November 2003

To understand the molecular basis of parasite‐specific anergy in human lymphatic filariasis caused by the nematode Wuchereria bancrofti, parasite antigen‐dependent cellular proliferation and cytokine gene expression were investigated. By reverse transcriptase polymerase chain reaction (RT‐PCR), the levels of cytokine mRNA were determined in the peripheral blood mononuclear cells (PBMCs) of different clinical groups of filariasis patients. This includes individuals with circulating microfilariae (MF), patients with chronic lymphatic obstruction (CP), and exposed but uninfected individuals (EN). Those with CP exhibited both a Th2 and a Th1 parasite antigen‐driven response. In PBMCs from those with MF, there was a marked downregulation of cellular response to parasite antigens, with lowered expression of Th1‐specific cytokines (IFN‐γ and IL‐2) and this was paralleled by increased IL‐10 expression. The EN individuals had a purely Th1‐type pattern with absence of IL‐4 and IL‐5 expression. Further, the mRNA expression of the costimulatory surface marker, CD80 (B7‐1), was not associated with either disease status or IL‐10 expression. There was a significant negative correlation between IL‐10 mRNA expression and PBMC proliferation in the MF individuals, thus indicating the possible role of IL‐10 in antigen‐specific hyporesponsiveness.
Elimination of P. berghei liver stages is independent of Fas (CD95/Apo‐I) or perforin‐mediated cytotoxicity
Authors: RENGGLI J. , HAHNE M. , MATILE H. , BETSCHART B. , TSCHOPP J. , CORRADIN G.
Published: November 2003

Immunization of mammals with irradiated malaria sporozoites protects from a subsequent contact with the parasite. Protective immunity is directed against the pre‐erythrocytic stages of the parasite, sporozoites and liver stages. Specific antibodies neutralize part of the infectious sporozoites injected by the mosquito vector, while liver stages are the target of a cellular immune response which is mediated by T cells. In this study, we evaluated the T‐cell dependent protection induced by the injection of P. berghei irradiated sporozoites and the contribution of perforin and of the receptor/ligand system CD95/CD95L, two T cell‐dependent mechanisms known to mediate elimination of target cells. Wild type, perforin deficient, CD95 mutant, CD95L mutant and perforin deficient/CD95L mutant mice were immunized with P. berghei irradiated sporozoites and submitted to a challenge with infectious sporozoites. All mice immunized with P. berghei irradiated sporozoites were protected against a sporozoite challenge, including perforin deficient/CD95L mutant animals. These results indicate that T cells do not kill malaria‐infected hepatocytes via one of the known pathways, but rather that activated parasite‐specific T cells produce cytokines which activate in cascade other mechanisms responsible for the intracellular elimination of the parasite.
Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys
Authors: Herrera Sócrates , De Plata Cecilia , González J. Mario , Perlaza Blanca Liliana , Bettens Florence , Corradin Giampietro , Arévalo‐Herrera Myriam
Published: November 2003

Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B‐cell epitopes recognized by human individuals. Three T‐cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS (p11‐p17) protein were used in ELISA assays to map the B‐cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T‐cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T‐lymphocyte proliferation and in vitroγ‐IFN production were evaluated. Two MAPs containing the same B‐cell epitope and either a promiscuous CS‐protein derived T‐cell epitope (p25) or the tetanus toxin epitope (p‐tt30) proved to be the most immunogenic and induced high levels of anti‐peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or γ‐IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitroγ‐IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.
Modelling Trypanosoma congolense parasitaemia patterns during the chronic phase of infection in N’Dama cattle
Authors: AGUR Z. , MEHR R.
Published: November 2003

We reanalysed parasitaemia profiles of the trypanotolerant N’Dama cattle (Bos taurus), consecutively infected with the same four clones of Trypanosoma congolense. Our analysis shows that each individual parasitaemia is characterized by progressively longer intervals between parasites waves. This pattern is most visible during the chronic phase of infection. In addition, the last of the four infections had a significantly larger overall duration of inter‐wave intervals. We retrieved these patterns by numerical simulations of a mathematical model, which incorporates assumptions about the molecular basis of antigenic variation and about the anti‐parasitic major immune processes. Six potential factors that may determine parasitaemia pattern were studied: carrying capacity of the host environment, intrinsic growth rate of the parasite, affinity maturation of the immune response, immune cell birth and death rate, levels of antibodies to variant surface glycoprotein and levels of antibodies to invariant antigens. Our simulations suggest that the first five factors are not likely to determine the chronic phase parasitaemia pattern whereas the sixth one, namely, antibody response to invariant antigens, yielded profiles consistent with the experimental data. Being cumulative, the immune response to anti‐invariant antigens may be increasingly effective as infection proceeds and in successive infections. Comparisons between N’Dama and Zebu and between chronic and acute phases will be needed to make a statement on the role of this phenomenon in trypanotolerance.
Murine Th1 clone defines a 60 kD tachyzoite antigen of Toxoplasma gondii
Authors: REICHMANN GABY , DLUGONSKA HENRYKA , FISCHER HANS‐GEORG
Published: November 2003

Toxoplasma gondii‐specific murine CD4+ T cell clone 3Tx9 belongs to the Th1 subtype by virtue of secreting high levels of interleukin(IL)‐2, interferon‐γ and tumor necrosis factor without producing IL4 and IL10. To characterize the clonal antigen, Toxoplasma lysate was separated by SDS‐PAGE and probed in T cell blot analysis with 3Tx9 T cells, revealing a fraction of about 60 kD molecular weight. This fraction proved highly stimulatory also for CD4+ splenocytes isolated from infected mice. The expression pattern of the relevant 60 kD antigen was determined by challenge of clone 3Tx9 with T. gondii strains from all three intraspecies subgroups and tachyzoites versus bradyzoites isolated from two strains as a source of antigen. While the T cell clone reacted with tachyzoites of all strains tested, bradyzoites lacked antigenic activity. Parallel T cell blot and ELISA confirmed co‐migration of the T cell‐stimulatory antigen p60 and rhoptry proteins ROP1, ROP2,3,4, and ROP5 among which ROP1 is a molecule of similar size and has only been shown on tachyzoites. However, a ROP1 knock‐out Toxoplasma mutant still had antigen activity for 3Tx9 T cells. Since the two known tachyzoite‐specific proteins, surface antigens SAG1/p30 and SAG2/p22, have a much lower molecular weight, we suggest that p60 represents a new T. gondii tachyzoite marker which is defined by clone 3Tx9.
Effect of praziquantel and oxamniquine treatment on human isotype responses to Schistosoma mansoni: elevated IgE to adult worm
Authors: WEBSTER M. , FALLON P.G. , FULFORD A.J.C. , BUTTERWORTH A.E. , OUMA J.H. , KIMANI G. , DUNNE D.W.
Published: November 2003

Pre‐ and post‐treatment antibody isotype responses to Schistosoma mansoni adult worm and soluble egg antigens were compared in a study population previously used to show that IgE against adult worm correlates negatively with intensity of reinfection following chemotherapeutic cure. IgG subclass responses to adult worm were lower after treatment whereas IgM and IgE were higher. The increase in IgE to adult worm was observed with different preparations of adult worm, including the worm tegument, and with both praziquantel and oxamniquine therapy. No significant difference was observed between pre‐ and post‐treatment isotype responses to egg antigens following either praziquantel or oxamniquine therapy.