Evaluation of two carrier protein-angiotensin I conjugate vaccines to assess their future potential to control high blood pressure (hypertension) in man.

AIMS: We aim to modulate the renin-angiotensin system (RAS) by active immunization against angiotensin I hormone (AI), potentially providing a novel conjugate vaccine treatment for hypertension in man. METHODS: Immunization studies in rat and human subjects compare the effectiveness of tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) vaccines for immunotherapy following conjugation with an AI peptide analogue (AI). Cardiovascular responses were assessed in immunized rats and human subjects (two-dose trial only), following increasing i.v. infusions of either AI or angiotensin II hormone (AII). RESULTS: The AI-TT and AI-KLH conjugate vaccines induced an equivalent immune response, and inhibition of the pressor effects to exogenous AI in rats. Single-dose clinical trials with both conjugate vaccines only resulted in an immune response to the KLH carrier protein. A two-dose clinical trial of AI-KLH conjugate vaccine resulted in a significant immune response to AI. A shift in diastolic blood pressure (DBP) dose-response was demonstrated following challenge with AI and AII for the study volunteer showing the largest anti-AI IgG induction. CONCLUSION: KLH was shown to be a suitable alternative to TT as a carrier protein for AI, thus supporting continued evaluation of our AI-KLH conjugate vaccine for treatment of hypertension in man.

Minor components in the sex pheromone of legume podborer: Maruca vitrata development of an attractive blend.

The legume podborer, Maruca vitrata (syn. M. testulalis) (F.) (Lepidoptera: Pyralidae) is a pantropical pest of legume crops. Sex pheromone was collected by gland extraction or trapping of volatiles from virgin female moths originating in India, West Africa, or Taiwan. Analysis by GC-EAG and GC-MS confirmed previously published findings that (E,E)-10,12-hexadecadienal is the most abundant EAG-active component with 2-5% of (E,E)-10,12-hexadecadienol also present. At least one other EAG response was detected at retention times typical of monounsaturated hexadecenals or tetradecenyl acetates, but neither could be detected by GC-MS. Laboratory wind-tunnel bioassays and a field bioassay of blends of (E,E)-10,12-hexadecadienal with (E,E )-10,12-hexadecadienol and a range of monounsaturated hexadecenal and tetradecenyl acetate isomers indicated greatest attraction of males was to those including (E,E)-10,12-hexadecadienol and (E)-10-hexadecenal as minor components. In subsequent trapping experiments in cowpea fields in Benin, traps baited with a three-component blend of (E,E)-10,12-hexadecadienal and these two minor components in a 100:5:5 ratio caught significantly more males than traps baited with the major component alone, either two-component blend, or virgin female moths. Further blend optimization experiments did not produce a more attractive blend. No significant differences in catches were found between traps baited with polyethylene vials or rubber septa, or between lures containing 0.01 and 0.1 mg of synthetic pheromone. Significant numbers of female M. vitrata moths, up to 50% of total catches, were trapped with synthetic blends but not with virgin females. At present there is no clear explanation for this almost unprecedented finding, but the phenomenon may improve the predictive power of traps for population monitoring.

Active immunization with angiotensin I peptide analogue vaccines selectively reduces the pressor effects of exogenous angiotensin I in conscious rats.

1. Male, Sprague-Dawley rats were actively immunized with novel angiotensin vaccines, and their pressor responses to exogenous angiotensin I (AI) and angiotensin II (AII) were assessed in vivo. Serum antibody titres were also measured. 2. The most effective vaccine consisted of an AI analogue conjugated with a tetanus toxoid carrier protein and adjuvanted with aluminium hydroxide. When this vaccine was injected on days 0, 21 and 42, pressor responses to AI on day 63 were significantly inhibited (maximum, 8.9 fold shift), but responses to AII were unaffected. The anti-angiotensin antibody titre was increased 32,100 fold, and, uniquely, these antibodies also cross-reacted with angiotensinogen. 3. These findings indicate that active immunization against AI may be a useful approach for treating cardiovascular disorders involving the renin-angiotensin system.

Clinical and parasitological responses after up to 6.5 years of ivermectin treatment for onchocerciasis.

There are plans to use mass treatment with ivermectin to clear all Africa of the worst ocular and cutaneous effects of onchocerciasis. However, there remains uncertainty about the most suitable treatment regimen and the likely effects of ivermectin on onchocercal skin disease. We have followed 948 subjects for over 6 years in a double-blind, randomized, controlled study of ivermectin for onchocerciasis in a hyperendemic focus in Sierra Leone. Using an intention-to-treat analysis we found a microfilarial prevalence of 16% 6 months after up to 4 annual doses of ivermectin, and 13% prevalence in the group receiving up to 10 doses of ivermectin at 6-monthly intervals. Microfilarial loads were well suppressed in both groups, but repopulation data suggest that adult female worms are still alive and fecund, strongly underlining the need to continue treatment. A clear effect of ivermectin was demonstrated on itching, with about one-third of cases being alleviated. Significant reductions in the prevalence of serious hyperkeratosis, and possibly dyspigmentation (leopard skin), were noted, but not for any other onchocercal skin lesion. Six-monthly and annual treatment regimens with ivermectin were equally effective in terms of dermatological and parasitological impact.

Viability and fertility of adult Onchocerca volvulus after 6 years of treatment with ivermectin.

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.

Endoplasmic reticulum protein expression in recombinant NS0 myelomas grown in batch culture.

During batch growth of two recombinant NS0 myelomas, an increase in the expression of the endoplasmic reticulum (ER) proteins (GRP78/BiP, GRP94, and ERp72) was observed. A marked increase in these proteins was associated with the decline phase of growth, an increase in the production rate of chimeric antibody, and a marked slowing or halt in the uptake of glucose and glutamate. Refeeding with glucose, glutamate, or a mixture of amino acids just prior to the onset of decline phase failed to repress induction. Although refeeding with glutamate led to an increase in specific productivity, there was no significant difference in the pattern of ER protein induction. These results indicate that an increase in ER protein expression is not solely related to productivity but also to certain changes that occur during the course of batch growth.

The effects of multiple doses of ivermectin on ocular onchocerciasis. A six-year follow-up.

BACKGROUND: Ivermectin has been shown to be a safe treatment for onchocerciasis and is now being distributed through mass treatment programs. Previous studies of up to 3 years of treatment have demonstrated that ivermectin improves anterior segment lesions and reduces the incidence of optic atrophy. The benefit of multiple doses of ivermectin on visual acuity and chorioretinitis has yet to be shown. METHODS: A community-based, double-blind, randomized, controlled trial of ivermectin was started in Bo, Sierra Leone, in 1987. Two cohorts are reported in this study: (1) 214 subjects had received four 6-month doses of ivermectin followed by up to six additional 6-month treatments. The second cohort, with 185 subjects, had received four 6-month doses of placebo followed by up to four annual doses of ivermectin. All subjects received a full ophthalmic examination in 1989 and again in 1994. For both cohorts, there was an 18-month gap between the fifth and sixth rounds of treatment. RESULTS: There was no significant difference in the prevalences of any ocular lesion nor of visual acuity categories between the cohorts at the second examination. Comparisons of the prevalences of anterior segment lesions for both cohorts combined between the first and second examinations show highly significant improvement (P < 0.001) for all lesions. Posterior segment lesions show a more variable pattern, with chorioretinitis showing highly significant deterioration (P < 0.001) and the emergence of new lesions in both groups. CONCLUSION: The authors conclude that annual treatment with ivermectin is effective in controlling ocular onchocerciasis apart from chorioretinal lesions and that a 6-month treatment gives no additional benefit.

A community trial of ivermectin for onchocerciasis in Sierra Leone: compliance and parasitological profiles after three and a half years of intervention.

We have studied the compliance patterns and the long-term effects of repeated ivermectin at various dosing intervals in a randomized controlled trial. The setting for the trial was six neighbouring communities hyperendemic for onchocerciasis in southern Sierra Leone. A total of 335 subjects attended a survey 18 months after the fifth treatment round. Of those randomized to ivermectin, over 85% had received at least three doses. There was no evidence that women of childbearing age were consistently under-treated, despite the criteria for exclusion from treatment. An intention-to-treat analysis showed that a 6-monthly ivermectin treatment regime satisfactorily suppressed microfilarial loads. Microfilarial repopulation was significantly slower over an 18-month period after multiple doses compared to a single dose. Further analysis of microfilarial repopulation suggests that there is a cumulative suppressive effect after at least the first three doses of ivermectin, and that an annual treatment interval is as effective for short-term microfilarial suppression as a 6-monthly interval.

How long do the effects of ivermectin on adult Onchocerca volvulus persist?

The persistence of the effects of ivermectin on the viability, morphology and reproductivity of adult Onchocerca volvulus was examined eighteen months after treatment with a single or five six-monthly doses of ivermectin and compared with untreated controls. Treated nodules were removed from patients participating in a randomised controlled trial of ivermectin in Sierra Leone. Adult filariae, 545 females and 348 males, were isolated by collagenase digestion. The nodules were significantly smaller, contained fewer young worms and supported lower microfilarial production in those treated with five doses of ivermectin. The productivity index, a measure of the reproductive potential of a worm population, was still reduced by 83% eighteen months after five doses and by 63% after a single dose compared to controls. These results show that worms recover their fertility even after multiple doses of ivermectin, but do so slowly compared to standard dosage intervals. In addition ivermectin may have a partial chemoprophylactic effect which contributes to the maintenance of low microfilarial production in conditions of on-going transmission.

Immunoaffinity chromatography in biorecovery: an application of recombinant DNA technology to generic adsorption processes.

The constant region of human kappa light chain (Ck) was linked to Escherichia coli beta-galactosidase, using standard molecular cloning techniques. The binding of Ck-beta-galactosidase fusions to a number of different murine monoclonal antibodies, specific for Ck, was improved by the insertion of spacers between Ck and beta-galactosidase: a cleavable linker was then introduced. Over-expressed Ck-beta-galactosidase fusion protein was purified using monoclonal antibodies immobilised on Sepharose 4B. Elution conditions were found that maintained beta-galactosidase activity so purified enzyme could be released on breaking the cleavable linker. A number of practical problems associated with maintaining stable fusion proteins and immunoaffinity column performance were identified.

Ivermectin does not reduce the burden of itching in an onchocerciasis endemic community.

Degrees of itching were estimated before and for 6 months after a fourth dose of ivermectin or placebo was given to 97 subjects in Sierra Leone. There was no reduction in itching attributable to ivermectin at any stage, but there were non-significant increases in the prevalence, severity and localization of itching within the first 2 months after ivermectin compared to placebo. We also found that cell-mediated immune responses to Onchocerca volvulus were significantly increased 4 weeks after a single dose of ivermectin compared to before treatment. A temporary reversal of the state of immunosuppression in people with onchocerciasis may counterbalance the reduction in skin microfilarial loads following ivermectin, with no consequent reduction in itching. The lack of effect of ivermectin on itching, a major symptom of onchocerciasis, while disappointing, need not detract from the success of mass distribution programmes.

Immunological studies on onchocerciasis in Sierra Leone. 2. Cell-mediated immune responses after repeated treatment with ivermectin.

Cell mediated responses of peripheral blood lymphocytes were tested by incorporation of I-125 dideoxyuridine in a standard proliferation assay. The cells were collected from age and sex-matched patients selected on the basis of their clinical and parasitological status. These patients were treated with either ivermectin or placebo at 6-monthly intervals for two years. For the purpose of comparison, the patients were divided into three groups: children aged 5-9 years; 10-19 year olds; and, those older than 20 years. Repeated drug treatment over the two year period had no effect on either mitogenic or non-parasite antigen (PPD) responses in a majority of individuals. However, in younger children (5-9 years) repeated treatment with ivermectin resulted in some enhancement of Onchocerca-specific responses measured 6 months after administration of the drug. This effect was more pronounced in children presenting with positive skin-snips. An age related decline in mitogenic responses was observed.

A community trial of ivermectin for onchocerciasis in Sierra Leone: adverse reactions after the first five treatment rounds.

We have studied the adverse reactions reported after ivermectin in 1745 villagers in southern Sierra Leone, allocated at random to receive ivermectin or placebo and treated 'double-blind' with 4 doses at six-monthly intervals. Six months after the fourth dose all eligible villagers received ivermectin regardless of their previous treatment. At the first treatment round more adverse reactions were reported by villagers treated with ivermectin than by those who received placebo. Reactions occurred most often on the second day after treatment. There were significant correlations between an individual's skin microfilarial load and the risk of developing adverse reactions. On re-treatment there was no significant excess of reported adverse reactions in the ivermectin group compared to the placebo group. Unlike other adverse reactions, the risk of cutaneous reactions after the first dose of ivermectin was not correlated with skin microfilarial load. In addition, after re-treatment with ivermectin, cutaneous reactions were reported significantly more often than with placebo. We confirm that ivermectin is safe for mass distribution, but adverse reactions should be monitored and treated after the first dose. Throughout this study ivermectin was well tolerated, with significantly more villagers returning for re-treatment after ivermectin than placebo, and all adverse reactions were self-limiting or successfully managed with symptomatic treatment. We question whether strict clinical monitoring should be routine at re-treatment, when only cutaneous reactions were consistently reported. If clinical monitoring could be used more selectively, distribution campaigns might be easier to manage and more cost-effective.

Partial characterization of 2-beta-mercaptoethanol-soluble surface-associated antigens of Onchocerca volvulus.

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-beta-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-beta-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15,000, 18,000, 28,000, 78,000, 98,000, 120,000 and 200,000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28,000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-beta-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

A community trial of ivermectin for onchocerciasis in Sierra Leone: clinical and parasitological responses to the initial dose.

A double-blind placebo-controlled trial of ivermectin was started in 1987 in 6 villages in southern Sierra Leone. 1625 villagers, 93% of the total population, were surveyed before treatment and allocated at random to the trial. Onchocerciasis was hyperendemic and of moderate intensity in the area. Typical onchocerciasis skin lesions were seen in most cases; the blindness rate was 1.5% and a further 4.3% had visual impairment. Six months after treatment 988 subjects (80%) were reassessed and microfilarial loads in the ivermectin group were found to be 10% of control levels. Additionally, blood eosinophil concentrations were reduced by one-quarter. The severity, but not the prevalence, of skin lesions was significantly reduced in the ivermectin group, with a particularly marked effect on papular eruptions. There had been no reduction in the prevalence of itching, nor had markers of general health shown improvement after ivermectin. Ivermectin is an effective microfilaricidal agent and may improve Onchocerca-related skin lesions after a single dose. However, the lack of obvious benefit to a target population after the first dose of ivermectin may reduce compliance with subsequent doses. This has implications for planned mass treatment initiatives in onchocerciasis endemic regions.

Immunological studies on onchocerciasis in Sierra Leone. 1. Pretreatment baseline data.

Cellular immune responses were tested in vitro using peripheral blood mononuclear cells from 203 individuals resident in an area of Sierra Leone where onchocerciasis is hyperendemic, and 32 individuals (Gambians) with no history of contact with Onchocerca volvulus. Mean reactivity to the mitogen, Concanavalin A, did not differ between these two groups, but responses to PPD were markedly lower in those with onchocerciasis. Proliferative responses to adult female O. volvulus antigen in the latter group were generally low although elevated reactivity was found in certain sub-groups. Higher responses were evident in infected 10-14 year olds, and there was an association between elevated reactivity to O. volvulus antigens and acute reactive dermatological signs, with individuals in the latter group also carrying higher dermal microfilarial loads. A sub-group presenting with lymphadenopathy showed the strongest associations of these three parameters. These results suggested the requirement for a threshold density of dermal microfilariae for induction of acute reactivity. The presence of immunosuppressive factors in soluble O. volvulus antigen was indicated by the ability to suppress, at low concentrations, the cellular responses to PPD of a proportion of individuals.

Isolation and in vitro translation of Onchocerca volvulus mRNA.

Adult Onchocerca volvulus recovered for excised nodules by dissection or treatment with collagenase have been used as a source of RNA for in vitro translation experiments. RNA was purified using either the hot phenol/SDS procedure or the guanidine isothiocyanate protocol. Immunoprecipitation experiments performed on in vitro products demonstrate a marked heterogeneity in responses by individed human infection sera. Further immunoprecipitation experiments demonstrate cross reactivity between O. volvulus and other filarial nematodes.

Acute illness in infants: a general practice study.

The character and frequency of acute illness in infants presenting to a general practice over a 16-week period was studied. Symptoms were classified as 'major' or 'minor' in accordance with the definitions used in a multicentre study in infant mortality.Of the 126 consultations reviewed, 106 (84 per cent) included at least one major symptom. None of the illnesses resulted in hospital admission or had a fatal outcome. It was concluded that this classification of symptoms into ;major' and ;minor' categories is not sufficiently discriminating to use in general practice. More specific definitions are required.

Mothers' appreciation of their children's symptoms.

A prospective study of mothers' reactions to their babies' symptoms was carried out in Newcastle upon Tyne. Forty-four mothers kept a health diary about their first infant, recording symptoms that occurred and action taken. Symptoms, most of which were minor, were present on three out of four days. Mothers were therefore making almost daily decisions about their babies' health, usually without medical advice. No evidence was found of mothers failing to appreciate the severity of symptoms or failing to seek medical help. Our findings reinforce those of other studies that parents are able to recognize their babies' illnesses, even in their first children.