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Aims: Current treatment for primary central nervous system lymphoma (PCNSL) involves high-dose methotrexate (HDMTX) with or without radiotherapy. Many published studies describing this approach include a highly selected group of patients. We report a single-centre experience of unselected cases of PCNSL., Materials and Methods: We retrospectively reviewed the case notes of 55 consecutive patients diagnosed with biopsy-proven PCNSL between 1995 and 2003 at Addenbrooke's Hospital Cambridge, UK. We describe the treatment and outcome, including survival, treatment-related toxicity and long-term functional disability., Results: At diagnosis, 45% of patients were considered unfit to receive treatment with HDMTX, owing to poor performance status or comorbidity. These patients had a median survival of 46 days and may not have been included in other published studies. The remaining patients were treated with a chemotherapy regimen, which included HDMTX. Patients who received at least one cycle of a chemotherapy containing HDMTX had a median survival of 31 months. Forty per cent did not complete planned chemotherapy owing to toxicity, disease progression or death. The median survival of patients treated with HDMTX aged 60 years compared with patients aged under 60 years was 26 months vs 41 months (P = 0.07), respectively. Younger patients treated with HDMTX, who achieved complete remission with chemotherapy, had a median survival of 56 months. We identified a high incidence of functional disability among survivors, resulting from a combination of the tumour itself, the neurosurgical procedure required for diagnosis and the late neurotoxicity of combined chemoradiotherapy., Conclusion: The treatment of PCNSL is associated with significant early and late toxicity. Further attempts to improve treatment should address mechanisms to reduce this toxicity. In particular, the benefit of radiotherapy in patients who achieve complete remission with HDMTX will remain uncertain until it is addressed in a multicentre, randomised trial.

BACKGROUND: The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding. METHODS: We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control. RESULTS: Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis. CONCLUSION: DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.

The time-course of the humoral immune response of female blackflies after a challenge with bacteria, different Onchocerca microfilariae species, bacterial endotoxin and microfilarial extract was investigated. Strong bacteriolytic and growth inhibition activities against the Gram-positive bacterium Micrococcus luteus were induced by all agents. Specific differences were found in activity levels and time-course. Notably the endotoxin lipopolysaccharide (LPS) induced a very early, profound bacteriolytic and antibacterial response, which declined within a day after injection. In contrast, the bacteriolytic activities after Escherichia coli D31 and Onchocerca microfilariae infections were lower, but remained elevated over the observation period of 4 days. The bacteriolytic activity was correlated to a haemolymph protein with a molecular weight of around 14 kDa. Anti-Gram-positive activity in the E. coli infected group appeared within the first 6 h. However, it took 4 days in the microfilarial infected blackflies to reach significant levels. The active agent was identified to be a peptide with a molecular weight of around 4-4.5 kDa. Activity against the Gram-negative bacteria E. coli was detected in blackflies injected with E. coli D31, O. dukei microfilariae and microfilarial extract on days 1 and 4 after injection. The immune response in S. damnosum s.l. naturally infected via a bloodmeal on cattle supported the findings of the experimental infections. Similarities of the immune response kinetics between bacterial and filarial infections suggested that intracellular Wolbachia bacteria, released from microfilariae, could be responsible for the antibacterial response. This is supported by the observation that the induction of an immune response in the Drosophila melanogaster mbn-2 cell line by the filarial extract is blocked by polymyxin B, which forms inactive complexes with bacterial LPS.

The FAR proteins of nematodes are small ( approximately 20 kDa), helix-rich, fatty acid and retinol-binding (FAR) proteins that appear to be confined to nematodes. We have carried out a comparative sequence and biochemical analysis of selected FAR proteins often species of filarial parasites (from the genera Onchocerca, Brugia, Wuchereria, Loa, Acanthocheilonema and Litomosoides). The sequences fall into two main groups corresponding broadly to the onchocercal and lymphatic filariasis parasites, and only those with unsheathed microfilariae were found to produce glycosylated FAR proteins. The proteins were released into culture medium by all the species and developmental stages investigated. Recombinant forms of two of these proteins (Ov-FAR-1 from O. volvulus and Bm-FAR-1 from B. malayi) were compared for ligand binding in fluorescence-based assays. Both were found to bind all-trans-retinol, (dansylamino) undecanoic acid (DAUDA), and oleic acid by competition. Both produced an identical, and dramatic, blue-shift in the fluorescence emission of DAUDA (from 541 to approximately 483 nm), indicative of similarity in the binding site environments of the two proteins. These findings indicate that there is strong conservation of the biochemical activities of the FAR proteins between the different parasite species, although they appear to have different post-translational modifications which may relate to the biology of the larvae.

A new family of fatty acid- and retinoid-binding proteins has recently been identified in nematodes. These are apparently nematode specific and have very different structures and binding characteristics to their mammalian counterparts. Retinoids have important roles in vision, tissue differentiation and repair, and can profoundly affect collagen synthesis. Binding proteins released by a parasite might therefore play a part in the generation of the skin and eye pathology seen in river blindness. They might also be involved in the formation of the subcutaneous nodules induced by this parasite.

Injection trials with compatible and non-compatible Onchocerca species into S. damnosum s.l., the vector of human and bovine onchocerciasis, demonstrated that the rapid killing of microfilariae within the blackfly's haemocoel is species specific. In the presence of the peptide RGDS as a blocking agent for integrin-like receptors of haemocytes, the survival of O. ochengi microfilariae in its natural intermediate host was significantly increased. This increased survival 24 h p.i. correlated with a significant decrease of apoptosis levels in the microfilariae following a 2 h exposure to the haemolymph in vivo. These findings suggest that haemocytes are directly involved in the killing of Onchocerca microfilariae in the blackfly.

Ov20 is a structurally novel 20-kDa retinol binding protein secreted by Onchocerca volvulus. Immunological and biological investigation of this protein has been hampered by the inability to maintain O. volvulus in a laboratory setting. In an effort to find a system more amenable to laboratory investigation, we have cloned, sequenced, and expressed cDNA encoding homologues of Ov20 from two closely related filarial species, Brugia malayi (Bm20) and Acanthocheilonema viteae (Av20). Sequence comparisons have highlighted differences in glycosylation of the homologues. We present here an analysis of mouse immune responses to Ov20, Bm20, and Av20. The results suggest a strong genetic restriction in response to native Bm20 that is overcome when recombinant, nonnative material is used. Reactivity of human filarial sera to the three recombinant proteins confirmed previous specificity studies with Ov20 but highlighted important differences in the reactivity patterns of the O. volvulus and B. malayi homologues that may be due to differences in glycosylation patterns. Ov20 is a dominant antigen in infected individuals, while Bm20 is not. The availability of the B. malayi homologue enabled us to use defined murine reagents and inbred strains for genetic analysis of responsiveness in a way that is not possible for Ov20. However, the close sequence similarity between Ov20 and Av20 suggests that the A. viteae model may be more suited to the investigation of the biological functions of Ov20.

A recombinant cysteine protease inhibitor, onchocystatin of the parasitic nematode Onchocerca volvulus, was tested for its role in microfilarial development in the simuliid vector. Onchocystatin was found to be present in female adults and skin microfilariae of the bovine parasite O. ochengi, the closest relative of O. volvulus. In addition the inhibitor could be detected as an excretory-secretory (E-S) product of the microfilariae. Co-injection of onchocystatin and the O. ochengi microfilariae into the surrogate vector Simulium ornatum s.l. significantly enhanced the recovery rates of the parasite within 24 h into the infection (P > 0.001). The findings suggest a possible role of onchocystatin in the evasion by the parasite of the immune response of its vector.

Co-injection of the parasite Onchocerca ochengi and the caspase inhibitors z-VAD.fmk and boc-D.fmk into the natural vector Simulium damnosum s.l. led to significantly increased survival of the parasites. Subsequent in situ apoptosis detection assays demonstrated that in the case of boc-D.fmk the enhanced survival was due to a diminished apoptosis level of the microfilariae in vivo. Additional assays using O. ochengi microfilariae which were coinjected with serine protease inhibitors into S. damnosum s.l. revealed that certain serine protease inhibitors can reduce the level of apoptosis.

Phenoloxidase (PO) is the key enzyme for melanin synthesis and plays an important role in the defense and recognition of pathogens in insects and other arthropods. We now report the upregulated transcription of the gene encoding the precursor of PO, prophenoloxidase, in Onchocerca-infected Simulium damnosum s.l., the main vector of human and bovine onchocerciasis in subsaharan Africa. Using homology-based generic primers in a polymerase chain reaction-based targeted differential display, the gene itself was identified and partially sequenced.

A range of protease inhibitors and carbohydrates were administered to the haemolymph of the vector Simulium damnosum s.l. to test for their effects on the success of an Onchocerca ochengi infection in vivo. We found that serine protease inhibitors led to a significant increase of parasite survival. Two sugars, D(+)-galactose and methyl-alpha-D-mannopyranoside, had the same effect. These effects are possibly due to the successful in vivo blocking of the two respective types of inducible immune molecules, the serine protease and the carbohydrate binding lectins, both of which have been identified in simuliids.

Onchocerca volvulus nodules were removed from 77 fully compliant patients in a longitudinal study of ivermectin treatment in Sierra Leone. The patients had participated in a randomized controlled trial and received either 4 annual doses of ivermectin or 10 6-monthly doses over 6 years. Worms were examined 9 months after the last treatment for evidence of changes in morphology, viability and reproductivity. The findings were compared with results for the 2 groups obtained at earlier surveys of the same study population. Repeated treatment at 6 and 12-month intervals has resulted in a marked ageing of the male worm population profile and a significant reduction in the proportion of live female worms found in the nodules. In addition, there has been a reduction in reproductivity of 90% or more. However, most of the worms found were still alive and potentially fertile, underlining the need for the continuation of regular ivermectin treatment to maintain the benefits achieved.

Vector-derived proteases are thought to be key to the regulation of filarial infections in Simulium damnosum s.I. To identify proteases of S. damnosum s.I. induced by infection with Onchocerca ochengi, a PCR-based differential display technique was used. By combining this method with homology-based serine protease primers transcripts can be detected from S. damnosum s.I. RNA.

Onchocerca ochengi, a bovine parasite, is a suitable model for research on human River Blindness. However, the microfilariae are normally found concomitantly with at least one of the other three bovine Onchocerca species O. dukei, O. gutturosa and O. armillata causing difficulties for the work on the microfilariae. We describe a simple and field applicable method for the separation of living O. ochengi microfilariae from the other Onchocerca species using Sephadex G-25 columns. Elution of mixed populations resulted in the passage of O. gutturosa and/or O. dukei in the initial 1 ml fraction with O. ochengi eluting as an almost 100% pure species in the 4th and 5th fractions.