Intrathecal 6-mercaptopurine: preclinical pharmacology, phase I/II trial, and pharmacokinetic study.

For over 30 years, oral 6-mercaptopurine (6-MP) has been a mainstay of systemic maintenance therapy for acute lymphoblastic leukemia. Despite its efficacy as an antileukemic agent, 6-MP has not been previously administered by the intrathecal (IT) route. In anticipation of a clinical trial of IT 6-MP, preclinical cytotoxicity and pharmacology studies were performed to define a safe, effective dose. The optimal concentration (greater than 1 microM) and duration of exposure (greater than 12 h) to 6-MP required for cytotoxicity were determined in vitro using human leukemia cell lines. The dose required to achieve the desired cerebrospinal fluid concentrations in humans was derived from pharmacokinetic parameters determined in rhesus monkeys. A phase I/II study was then performed in pediatric patients with refractory meningeal leukemia. Nine patients (aged 3.5 to 16 years) with chronic meningeal leukemia (2 to 6 central nervous system relapses) were entered onto the study. All had previously failed, at a minimum, IT methotrexate, IT cytarabine, and cranial (+/- spinal) radiation. A 10-mg IT dose of 6-MP (calculated to produce cytotoxic cerebrospinal fluid levels for 12 h) was administered twice weekly for 4 weeks. There were four complete responses and three partial responses. The duration of complete responses ranged from 7 to 22 weeks. Observed toxicities were not dose limiting and included mild headache (three patients) and minimal nausea (two patients). Pharmacokinetic studies performed in patients confirmed that cerebrospinal fluid concentrations of 6-MP were greater than 1 microM for 12 h. These results indicate that the IT administration of 6-MP is feasible, is not associated with significant toxicity, and has definite activity in patients with refractory meningeal leukemia.

Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus with homologues in other filarial nematodes and Caenorhabditis elegans.

The full-length cDNA corresponding to an Onchocerca volvulus antigen, OvMBP/11, which had been selected as a serodiagnostic tool was isolated, sequenced, and the native antigen encoded by the cDNA characterised. The cDNA encodes a protein of 20.5 kDa (termed Ov 20) containing a putative signal peptide. Southern blot analysis indicates that there is only a single O. volvulus gene corresponding to Ov 20 but it has significant sequence similarity to genes corresponding to two 20.5-kDa predicted proteins of Caenorhabditis elegans. Homologues of the Ov 20 gene were detected at high stringency by Southern blot in the other Onchocerca species O. gibsoni, and O. gutturosa and at lower stringency in the related filarial nematodes Brugia malayi and Acanthocheilonema viteae. The Ov 20 native antigen has two molecular mass forms, 20 and 22 kDa, in all the life cycle stages studied. These isoforms have different levels of N-linked glycosylation on a peptide backbone of 17.5 kDa. Immunolocalisation and in situ hybridisation studies demonstrated that Ov 20 is transcribed and translated in the body wall of adult females and also in microfilariae, third and fourth stage larvae. Antigen was detected in the supernatant of in vitro cultured adult female nematodes. The B. malayi and A. viteae homologues are antigenically cross-reactive to Ov 20, share the same size peptide backbone but differ in their degree of glycosylation.

A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens.

A specific serodiagnostic test for onchocerciasis has been a priority objective of the World Health Organization. Fragments of cDNA encoding Onchocerca volvulus antigens selected on the basis of their specificity for this parasite were subcloned into a protein purification and expression system. No individual recombinant antigen showed a high sensitivity in an enzyme-linked immunosorbent assay because of heterogeneity in the response of O. volvulus-infected individuals. However, a cocktail of three recombinant proteins showed 96% sensitivity with 100% specificity, compared with 99% sensitivity and only 59% specificity against a crude O. volvulus extract. The sensitivity of detection of individual antigens varied between sera taken from individuals from different geographic areas infected with O. volvulus, but when used as a cocktail, all but one of the microfilaria-positive individuals from all the geographic areas studied were detected. Recombinant probes provide a practical basis for specific diagnostic tests for helminth infections.

The effect of ivermectin treatment on the antibody response to antigens of Onchocerca volvulus.

The effect of the microfilaricidal drug ivermectin on the antibody response to a detergent extract of adult Onchocerca volvulus (OvAg) and a number of specific recombinant peptides was examined. Three of the peptides were combined in a serodiagnostic 'cocktail' and the effect of ivermectin on the diagnostic performance of this assay was assessed. Immunoglobulin (Ig) G1 serum levels in response to OvAg significantly decreased following ivermectin treatment. The antibody response to only one recombinant peptide (OvMBP29) was significantly affected, with IgG levels decreasing following treatment. Levels of total IgE increased following treatment. No correlation was observed between initial antibody level (or change in antibody level) and any adverse reaction to treatment. The serodiagnostic 'cocktail' was 100% sensitive before and after the use of ivermectin. A serodiagnostic assay using specific recombinant peptides can be used to evaluate infection in the absence of dermal microfilariae in areas where ivermectin is used.

Onchocerciasis hyperendemic in the Unturán Mountains: the value of recombinant antigens in describing a new transmission area in southern Venezuela.

A recently described hyperendemic onchocerciasis area, located in the Unturán Mountains (between the Siapa and Orinoco basins) of southern Venezuela was studied using a cocktail of 3 low molecular weight onchocercal recombinant antigens (OvMBP/10, OvMBP/11, and OvMBP/29). The resulting seroepidemiological data were compared with those from a hypoendemic community (Altamira) situated in the northern coastal mountain range. Parasitological (skin biopsy) and serological (enzyme-linked immunosorbent assay, ELISA) methods for the specific diagnosis of Onchocerca volvulus in these 2 very different endemic areas were, respectively, 88% and 96% sensitive in Unturán, and 57% and 91% sensitive in Altamira. The mean microfilarial load, the mean optical density (OD), and the seropositivity rates all increased significantly with age in both communities. The serological variables (mean OD and prevalence of anti-O. volvulus antibodies) were both significantly higher in Unturán than in Altamira for children and young adults (aged < 25 years), although above this age no differences between communities were detected. Seroprevalence had already reached 50% in the under 15 year-olds examined at Unturán but was just 5% at Altamira for the same age-class. The prevalence of specific antibodies (mainly a marker of exposure to risk of infection) exceeded 85% in the remaining age-categories at the hyperendemic area. This is in agreement with the high community microfilarial load recorded in Unturán (> 20 mf/mg) and the presence of sclerosing keratitis and hanging groin, suggesting that onchocerciasis is a public health problem in this community. The ELISA test used here, based on a cocktail of 3 low molecular weight onchocercal recombinant antigens, appears, therefore, to constitute a practical tool for the description of endemicity levels in remote areas, particularly given the fact that finger-prick blood samples are routinely taken from children in the Upper Orinoco region for surveys of malaria incidence. Such studies could aid in defining the true extent of the Amazon focus (still unknown) and providing priority indicators for the selection of communities where onchocerciasis control programmes should be implemented.

Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes that express iron-regulated proteins.

OBJECTIVE: To determine efficacy of intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membrane proteins (OMP) expressing iron-regulated OMP (IROMP) in conferring protection against experimental challenge exposure. ANIMALS: 52 male New Zealand White rabbits. PROCEDURE: Rabbits were vaccinated intranasally on days 0, 7, and 14; some vaccines included cholera toxin (CT) as an adjuvant. Concentrations of intranasal IgA and serum IgG antibodies against P multocida OMP were determined. In experiment A, rabbits were vaccinated with either phospate-buffered saline solution (PBSS), PBSS-CT, OMP-CT, or IROMP-CT, challenge-exposed intranasally on day 16, and euthanatized and necropsied on day 28. Rabbits were also vaccinated with OMP or IROMP without CT and were not challenge-exposed. In experiment B, rabbits were vaccinated with PBSS, PBSS-CT, IROMP, or IROMP-CT. On day 17, rabbits were challenge-exposed intranasally. Nasal bacteria and antibodies were determined on day 24. RESULTS: In experiment A, OMP-CT vaccination stimulated mucosal and systemic antibody responses to the bacterium and enhanced resistance against challenge exposure. Intranasal bacterial counts were not significantly reduced. Vaccination with IROMP-CT stimulated mucosal and systemic antibodies, enhanced resistance to challenge exposure, and significantly reduced nasal bacterial counts. In experiment B, natural infection was detected in several rabbits at challenge exposure; however, IROMP-CT-vaccinated rabbits had significantly higher serum and nasal antibody responses, compared with other rabbits IROMP-CT-vaccinated rabbits had significantly lower nasal bacterial counts compared to control rabbits. CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal vaccination of rabbits with P multocida outer membranes containing IROMP and CT stimulated immunity against experimental pneumonic pasteurellosis.

NF1 plexiform neurofibroma growth rate by volumetric MRI: relationship to age and body weight.

OBJECTIVE: To longitudinally analyze changes in plexiform neurofibroma (PN) volume in relation to age and body growth in children and young adults with neurofibromatosis type 1 and inoperable, symptomatic, or progressive PNs, using a sensitive, automated method of volumetric MRI analysis. METHODS: We included patients 25 years of age and younger with PNs entered in a natural history study or in treatment trials who had volumetric MRI over > or =16 months. RESULTS: We studied 49 patients (median age 8.3 years) with 61 PNs and a median evaluation period of 34 months (range 18 to 70). The PN growth rates varied among patients, but were constant within patients. Thirty-four patients (69%) experienced > or =20% increase in PN volume during the observation period. PN volume increased more rapidly than body weight over time (p = 0.026). Younger patients had the most rapid PN growth rate. CONCLUSIONS: Volume increase of plexiform neurofibromas is a realistic and meaningful trial endpoint. In most patients plexiform neurofibroma growth rate exceeded body growth rate. The youngest patients had the fastest plexiform neurofibroma growth rate, and clinical drug development should be directed toward this population. Age stratification for clinical trials for plexiform neurofibromas should be considered.

The effects of vector control on the antibody response to antigens of Onchocerca volvulus.

The effects of exposure to infective larvae on the antibody response to a cocktail of specific recombinant antigens of Onchocerca volvulus and to a worm extract were evaluated by comparing the responses of individuals from a vector controlled area with those from an area of continuing transmission by ELISA. Individuals from the vector controlled areas were found to have reduced responses to both antigen preparations. A microfilerdermic (mf-) individuals from the area of vector control exhibited significantly lower total and subclass IgG responses to the worm extract. In contrast, the responses to the cocktail of specific recombinants were significantly reduced in individuals from the area of vector control who were still microfilerdermia positive (mf+). The distribution of IgG subclass specific responses was similar to both antigen preparations, both dominated by the IgG4 and IgG1 subclasses. IgG1 responses to the worm extract remained elevated in the vector controlled individuals but IgG4 was significantly reduced in the mf- individuals. Both subclasses reflected the total IgG response to the cocktail of recombinants and were significantly reduced in individuals from the vector controlled area, when compared to individuals from the hyperendemic area. IgG1 responses to the cocktail of recombinants are significantly lower than IgG4 in all individuals and virtually absent in individuals from the vector-controlled area. Measuring total IgG and IgG4 is more sensitive than IgG1 in detecting infection, 100 or 97% respectively, but they remain elevated in the individuals from the vector controlled areas even after 8-10 years interruption of transmission. These results have important implications for the serological monitoring of control programmes in individuals who have previously been infected.

Intraventricular concentration times time (C x T) methotrexate and cytarabine for patients with recurrent meningeal leukemia and lymphoma.

BACKGROUND: Intraventricular chemotherapy results in more uniform drug distribution within the subarachnoid space and allows for more flexible drug administration schedules. The authors report their experience with an intraventricular concentration times time (C x T) chemotherapy regimen for recurrent meningeal leukemia and lymphoma. METHODS: Twenty-one patients (median age, 11.6 years) received C x T therapy for meningeal acute lymphoblastic leukemia (n = 18), Burkitt's lymphoma (n = 2), or undifferentiated leukemia (n = 1). Prior therapy included standard intrathecal (IT) methotrexate and cytarabine, cranial or craniospinal radiation (median, 24 Gy), and 0-5 experimental treatment modalities. C x T induction therapy consisted of 2 mg of intraventricular methotrexate administered daily for 3 days every 10 days, for 4 courses. Patients were then consolidated with 4 courses of alternating intraventricular cytarabine (15 mg/day) or methotrexate (2 mg/day) daily for 3 days every 2 weeks (2 courses of methotrexate and 2 courses of cytarabine). Maintenance therapy consisted of alternating monthly courses of C x T methotrexate or cytarabine. RESULTS: Ninety-three percent of patients (14 of 15) who were evaluable for response achieved a complete remission in a median of 10 days (range, 2-40 days). Median remission duration was 15 months. Fourteen patients died of recurrent disease or systemic treatment-related complications; 2 patients are alive, off treatment, and in continuous complete remission for 59+ and 89+ months; 1 patient experienced a meningeal relapse at 24 months on C x T therapy but was reinduced with the C x T regimen, received craniospinal radiation, and is in remission at 142+ months; and 3 are alive with disease at 32+, 72+, and 81+ months. One patient was lost to follow-up. CONCLUSIONS: This regimen appears to be an effective and well-tolerated palliative treatment for patients with recurrent meningeal leukemia and lymphoma.

Heterogeneity of IgG antibody responses to cloned Onchocerca volvulus antigens in microfiladermia positive individuals from Esmeraldas Province, Ecuador.

The prevalence of IgG antibodies to three recombinant O. volvulus antigens, OvMBP/10, OvMBP/11 and OvMBP/29 was determined in a group of 94 microfilaria positive (mf+) individuals resident in the hyperendemic onchocercal area of Esmeraldas Province, Ecuador. Clone OvMBP/11 was the antigen most frequently recognized by patients sera followed by OvMBP/10 and OvMBP/29. When a cocktail of the three recombinant antigens was used the proportion of positive sera increased to 100%. Antibody responses to the fusion partner maltose binding protein (MBP) were low in comparison with those to the cloned antigens and no correlation of responses between individual antigens was observed. The relative level of antibody response to each of the clones in the cocktail varied between individuals. The distribution of IgG responses to OvMBP/11 was bimodal and those to OvMBP/29 and OvMBP/10 were positively and negatively skewed, respectively. When the three recombinant antigens were used in combination this variation was minimized and the pattern of responses showed a normal distribution as was also seen to crude O. volvulus antigen. The cocktail of recombinants thus offers excellent diagnostic sensitivity in combination with the parasite specificity demonstrated previously.