Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni.

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon gamma (IFN-gamma) synthesis by cells from vaccinated animals (7), suggested differential CD4+ subset stimulation by the different parasite stimuli. To confirm this hypothesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-gamma and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patently infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval antigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.

CD4+ T cell-mediated granulomatous pathology in schistosomiasis is downregulated by a B cell-dependent mechanism requiring Fc receptor signaling.

The effector functions of CD4+ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4+ lymphocytes, and previous studies have implicated CD8+ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell-deficient (muMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor-deficient (FcR gamma chain knockout) mice display the same exacerbated egg pathology as that observed in infected muMT mice, the B cell- dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4+ T cells.

Studies on the degradation of HeLa non-histone proteins.

The hydrolysis of HeLa non-histone nuclear proteins over 24 h has been monitored in dilute alkali at 4, 15 and 25 degrees C using the standard ninhydrin estimation, dansylation and various electrophoresis techniques. Under conditions (up to 0.2 N NaOH, 4 degrees C) that do not release a significant quantity of ninhydrin-positive material or new N-terminal end group considerable breakdown was observed by two-dimensional electrophoresis analysis. The number of stained spots decreased from approx. 140 to 25--30. No internal protease activity could be found. Labelling studies (14C-labelled amino acids) showed that much of the hydrolysed material was extracted from the gel during normal staining and destaining procedures. Peptides could be extracted from alkali-hydrolysed non-histone protein with acid/ethanol and could be further separated by thin-layer chromatography on silica gel G. Short-term labelling of HeLa cells (14C-labelled amino acids for up to 60 min) revealed that these peptides probably have a high rate of turnover. [14C]Glucosamine studies also indicated the presence of considerable carbohydrate material in the low molecular weight products of this alkaline hydrolysis. Various standard proteins and histones were unaffected by hydrolysis in up to 0.2 N NaOH (4 degrees C, 24 h) as judged by gel electrophoresis. Seven different phosphate-splitting enzymes and an esterase had no effect on the non-histone protein electrophoresis patterns but a preparation of phospholipase C which had no protease activity towards eight standard proteins did produce considerable breakdown in HeLa non-histone proteins similar to that produced by 0.2 N NaOH at 4 degrees C.

Genetic diversity of Schistosoma mansoni: quantifying strain heterogeneity using a polymorphic DNA element.

Intraspecific genetic variation among 14 geographic isolates of Schistosoma mansoni was quantified using a molecular marker to examine individual genotypes. Genetic crosses demonstrated maternal inheritance of S. mansoni DNA element pSM750. This element revealed diagnostic banding profiles, which allowed accurate strain identification. Most strains had similarity indices greater than 0.75 indicating that within-strain variation in these laboratory parasite populations was low. However, individual parasites from the NMRI strain were quite diverse (S = 0.40). Genetic heterogeneity among strains was quantified using a phenogram of mean genetic distance. Strain diversity between two geographic regions was quantified using a similarity index and was estimated to be substantial among isolates collected from a single local site.

A genomic change associated with the development of resistance to hycanthone in Schistosoma mansoni.

Ribosomal gene probes were used to investigate the genetic basis of drug resistance in schistosomes in a model where resistance to the anthelmintic hycanthone (HC) is generated by exposing immature worms to the drug. Two strains of Schistosoma mansoni, JHU and NMRI, were used. Drug resistance could be produced in the JHU strain by treatment with HC, but was also found to occur spontaneously. In contrast, it was not possible to detect or produce resistance to HC in the NMRI strain. A genomic alteration accompanied the development of resistance. The change was evidence by the occurrence of restriction fragment length polymorphisms (RFLPs) when Southern blots of genomic DNA from HC-resistant worms were hybridized with the ribosomal probe pSM389, which contains part of the small rRNA gene plus non-transcribed spacer (NTS) sequence. The most reliable marker of HC-resistance was a 3.6-kb BamHI fragment which was present and heritable in 7 drug-resistant lines derived from the JHU strain but absent from the parent JHU population and from NMRI parasites. The universal absence of the 3.6-kb RFLP in HC-sensitive individuals and its presence in the drug-resistant progeny suggest that resistance results from an induced change in the population rather than from selection of HC-resistant parasites. The rRNA gene sequence responsible for detecting the 3.6-kb RFLP appears to be localized either to the NTS or to the 5' end of the small rRNA gene, since hybridization to a probe containing sequence from the rRNA gene contiguous and downstream from the insert of pSM389 failed to reveal the RFLP. These results show that the development of resistance to HC is accompanied by a genomic rearrangement.

Molecular studies of Biomphalaria glabrata, an intermediate host of Schistosoma mansoni.

The freshwater gastropod Biomphalaria glabrata is one of the most important invertebrate hosts of the helminth parasite Schistosoma mansoni. Investigators are using different strategies to determine the molecular basis of this snail-parasite relationship. Of particular interest are the identification of parasite resistance genes in the snail, and the application of molecular probes to better understand the epidemiology of schistosomiasis. This review will focus on recent advances that have been made on genome analysis of B. glabrata. Much of this work has centred on the use of random amplification of polymorphic DNA-PCR-based technology, with restriction fragment length polymorphism analysis and the generation of expressed sequence tags from the snail. A brief discussion of how parasite products may complicate this analysis is also given, along with an indication of the scope of the problems that lie ahead.

Differential gene expression in haemocytes of the snail Biomphalaria glabrata: effects of Schistosoma mansoni infection.

Parasite encapsulation and destruction in Biomphalaria glabrata has been shown to involve the cellular component of the snail's internal defence system, the haemocytes. To identify genes involved in the immunobiology of these cells, we used the method of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) to investigate differential gene regulation in haemocytes isolated from Schistosoma mansoni exposed and unexposed snails. RNA isolated from circulating haemocytes from resistant snails (BS-90 stock), previously exposed to S. mansoni, was analysed using 12 different arbitrary primers in conjunction with an anchored Oligo d(T(11)CG) primer. Transcription profiles between haemocytes of parasite exposed and unexposed snails were compared and a total of 87 differentially regulated bands were identified and isolated. Of these, 65 bands were cloned and used as probes in Southern blots to show the presence of corresponding sequences in the snail genome. RT-PCR was performed to verify the regulation of these transcripts. DNA sequence analysis showed that the majority of the cloned sequences were novel, although a few showed a high degree of sequence similarity to other sequences in the DNA and protein databases. One of these included a differentially expressed transcript that showed a significant degree of sequence identity to E. coli transposase Tn5, an enzyme whose activity is normally associated with generating mobility and instability in the genome.

Application of 1 nm gold probes on paraffin wax sections for in situ hybridisation histochemistry.

An in situ hybridisation technique that uses 1 nm immunogold reagents and silver enhancement was devised to detect biotinylated DNA viral probes in formalin fixed, paraffin wax sections of human cervix. DNA probes labelled with biotin-11-deoxyuridine triphosphate were detected after hybridisation to nucleic acid sequences by an antibiotin antibody, followed by a gold labelled secondary antibody. Silver enhancement then permitted visualisation of the signal at the light microscopic level. The method was reliable and produced less background staining than previously described methods. The signal could be enhanced by epi polarisation microscopy. Furthermore, biotinylated DNA probes may be detected directly by a 1 nm gold labelled goat antibiotin antibody without loss of labelling intensity, and this may be preferable to the longer two layer technique, previously described.

In situ hybridisation for the identification of Helicobacter pylori in paraffin wax embedded tissue.

A method for identifying Helicobacter pylori using a non-isotopic in situ hybridisation technique is described. A probe generated by polymerase chain reaction (PCR) with primers directed against parts of the Helicobacter pylori 16SrRNA sequence was used. Paraffin wax embedded gastric biopsy specimens from patients with and without gastritis were hybridised with the probe, and the method was shown to be sensitive and specific for H pylori.

Laryngeal papillomatosis: correlation between severity of disease and presence of HPV 6 and 11 detected by in situ DNA hybridisation.

A technique using a biotin-streptavidin polyalkaline phosphatase complex was applied to routinely fixed and processed biopsy specimens of laryngeal papillomata from 45 patients taken over the past 20 years to detect human papilloma virus (HPV) types 6 and 11. Two thirds of both adult and juvenile onset cases were positive for HPV 6 or HPV 11 or both. Five specimens of normal vocal cord epithelium were negative for HPV 6 and 11. The detailed clinical history, endoscopic findings, success of treatment and eventual prognosis were compared with the HPV state of biopsy material for each patient. Patients with multiple confluent lesions when first seen, whose histology showed florid koilocytosis and who had strongly positive reactivity for HPV 6 or 11 present in the surface epithelial cell nuclei, had a poor prognosis requiring multiple endoscopies to control their disease.

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

Sensitive in situ hybridisation technique using biotin-streptavidin-polyalkaline phosphatase complex.

A sensitive in situ hybridisation technique, using a biotin-streptavidin-polyalkaline phosphatase complex detection system, was successfully applied to smears of fresh cultured cells, frozen sections, and formalin fixed paraffin processed tissue: the procedure was successful for DNA-DNA hybridizations using a variety of DNA probes. The detection method is rapid, reliable, and economical producing a purplish-blue precipitate at the site of hybridisation and clearly visible by low power light microscopy.

Demonstration of multiple HPV types in normal cervix and in cervical squamous cell carcinoma using the polymerase chain reaction on paraffin wax embedded material.

The prevalence of human papilloma virus (HPV) types 6, 11, 16 and 18 was investigated using the polymerase chain reaction on formalin fixed, paraffin wax embedded material in 19 cases of cervical squamous cell carcinoma and in 10 normal cervices. HPV DNA was detected in 16 of 19 carcinomas, with multiple types present in 11 of these. HPV 16 or 18, or both, were present in all cases in which HPV was shown. Six of 10 cases of normal cervix contained HPV; five of these contained two or more HPV types, including HPV 16 or 18, or both. This study shows the feasibility of using the PCR on paraffin wax embedded material and indicates a high rate of carriage of multiple HPV types in both normal and neoplastic cervix.

Demonstration of Epstein-Barr virus in primary brain lymphoma by in situ DNA hybridisation in paraffin wax embedded tissue.

Tumour tissue from 29 patients with primary brain lymphoma was reviewed to determine if there was an aetiological association between Epstein-Barr virus and polyclonal and monoclonal lymphoproliferations. The morphology and immunophenotype in 24 patients for whom paraffin wax embedded tissue was available were studied. A high grade pleomorphic tumour morphology with plasmacytoid features was seen in 13 tumours. Because of the large number of pleomorphic lymphomas, all tumours were examined for the presence of the Epstein-Barr virus genome using in situ DNA hybridisation. A panel of three biotinylated probes to different sequences in the Epstein-Barr virus genome was used. Positive hybridisation with one or more probes was shown in tumours from 11 patients. The remaining tumours gave no hybridisation signal. There was no correlation between positive hybridisation and morphological subtype or clinical outcome.

Identification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR.

AIMS: To determine the prevalence of Helicobacter pylori colonisation in the mouths of patients with H pylori gastritis. METHODS: A nested polymerase chain reaction test for the 16S ribosomal RNA gene of H pylori was used on saliva, dental plaque, gastric juice and gastric biopsy specimens from patients attending a dyspepsia clinic. RESULTS: Thirteen patients had histologically confirmed Helicobacter associated gastritis. Twelve of these had positive gastric aspirates by PCR. Five had at least one positive oral specimen. Eight patients with normal gastric biopsy specimens had no PCR positive oral specimens or gastric aspirates. All, however, had PCR positive gastric biopsy specimens. In an attempt to determine the origin of these positive results in normal patients, it was shown that biopsy forceps could contaminate specimens with DNA from previous patients. CONCLUSION: The demonstration of the organism in the mouths of a substantial proportion of dyspeptic patients has major implications for the spread of H pylori and identifies a potential source for reinfection following eradication of the organism from the stomach.

Epstein-Barr virus in normal, pre-malignant, and malignant lesions of the uterine cervix.

AIM--To detect the presence or absence of Epstein-Barr virus (EBV) in cervical lesions ranging from normality to invasive malignancy. METHODS--Eighteen randomly selected cases of invasive squamous cell carcinomas of the uterine cervix were examined as well as 25 cases each of normal cervices and those showing cervical intra-epithelial neoplasia (CIN) I, II, and III. DNA-DNA in situ hybridisation, using a biotinylated probe to the Bam H1 "W" fragment of EBV, was carried out in addition to the polymerase chain reaction using specific primer sequences that flank a 153 base pair segment of the Bam H1 "W" region of the EBV genome and which do not cross-amplify other DNA herpes viruses. Positive control material included paraffin wax embedded P3 HR1 lymphoblastoid cells (containing high copy numbers of EBV) and two nasopharyngeal carcinomas positive for EBV. RESULTS--Neither normal nor CIN I tissue was positive. Eight per cent of CIN II tissue was positive; 8% of CIN III, and 43% of carcinomas were positive for EBV. CONCLUSION--The study shows that the virus is present in some cases of cervical carcinoma and to a lesser degree in some premalignant lesions of the cervix, but the exact association between it and cervical oncogenesis, be it causative or incidental, remains to be determined.

PCR in situ hybridisation detection of HPV 16 in fixed CaSki and fixed SiHa cell lines.

AIMS: To investigate the feasibility of using fixed cells with the polymerase chain reaction (PCR) in situ hybridisation and to investigate possible reasons for reaction failure. METHODS: Fixed SiHa and CaSki cells were used in an experimental model of PCR in situ hybridisation for the detection of low and intermediate copy number viral infection in fixed cells. RESULTS: PCR in situ hybridisation was able to detect one to two copies of human papillomavirus (HPV) 16 in SiHa cells, using small fragment amplicons (120 base pairs), confirming the high detection sensitivity and flexibility of the technique. Problems were encountered with localisation of PCR amplified product in CaSki cells (200-300 copies of HPV 16 per cell) owing to diffusion of product post amplification. Overall, 40% of reactions were successful, which confirms the current unreliability of the technique. Within cell preparations, about 50% of cells contained amplified product. CONCLUSION: PCR in situ hybridisation represents the marriage of two revolutionary molecular pathological techniques. However, it is currently unreliable, with reaction failure common. Standardised, dedicated equipment is urgently required if the technique is to achieve universal acceptance. In the future, the technique may be used to detect chromosomal translocations in human tumours and to study cellular gene expression.

Regional and splenic lymphocyte proliferative responses of mice exposed to normal or irradiated Schistosoma mansoni cercariae.

Developing larvae of Schistosoma mansoni migrate through various tissues en route to the liver and mesenteric veins of their definitive host. Regional (lymph node) and systemic (spleen) blastogenic responses to cercarial, adult and egg antigens were measured in CBA/J mice at various times after exposure to normal or irradiated S. mansoni cercariae. Among the separate lymph node groups studied were those draining the tail, thoracic region, intestines, head and neck, and the pelvis. Blastogenic responses were assayed by a micromethod requiring 10(5) cells in 20 microliter volumes per culture. Up to 5 weeks post-cercarial exposure the pattern of responses in lymphoid tissues of infected mice coincided with the migratory route of the parasites. Following oviposition, cellular reactivity was pronounced in all lymph node groups. The reactivity of mice exposed to irradiated cercariae followed a pattern suggestive of a sustained antigenic stimulus only in the nodes draining the tail and lungs. Splenic (systemic) reactivity was roughly comparable between the two exposure groups. These data show the independence and vast differences in the host regional responses following normal or irradiated cercarial exposure.

Effect of a cryopreserved live vaccine on resistance in mice with a pre-existing Schistosoma mansoni infection.

Experiments were conducted to determine the effect of anti-schistosomal vaccine exposure on the level of pre-existing resistance in mice with a bisexual Schistosoma mansoni infection. C57BL/6 mice with light S. mansoni infections of 8 weeks duration were injected with 10 Krad-irradiated, cryopreserved and thawed schistosomules. Four weeks later the mice were exposed to normal cercariae, and adult worms collected by perfusion 6 weeks post-challenge. In 4 experiments, the baseline levels of resistance in infected mice ranged from 19% to 50% reduction in challenge worm burden (mean of 36%). Although vaccine administration slightly raised the overall level of resistance in infected mice (mean of 49%), in only 1 experiment was the increase over that in infected mice statistically significant. The levels of resistance attributed to the patent infection and vaccine exposure were not additive. Vaccine exposure had no effect on recovery of the adult worms of the primary infection.

Schistosoma mansoni: radiation dose and morphologic integrity of schistosomules as factors for an effective cryopreserved live vaccine.

The effectiveness of a cryopreserved, irradiated schistosomule vaccine against an homologous Schistosoma mansoni cercarial challenge was tested in C57B1/6 mice. Highly significant levels of protection developed consistently when mice were immunized with the vaccine irradiated at 10-20 Krad, i.e., doses below that considered optimal for irradiated cercariae (50 Krad). Cryopreserved schistosomules irradiated at 10 or 20 Krad induced greater levels of protection than did schistosomules irradiated at 2, 5, 30, or 50 Krad. Protective immunity developed as early as 3 weeks post-immunization. When immunizing inocula were injected at various times post-thaw, or when schistosomule subpopulations of normal-appearing, damaged or dead organisms were injected, those populations which had appeared to sustain the least degree of damage were those most capable of stimulating protective immunity. These findings highlight the hazards of extrapolating conditions considered standard for an irradiated cercarial vaccine to one in which cryopreservation, for storage of the schistosomules, is an added stress.