Characterization of an immunodominant Onchocerca volvulus antigen with patient sera and a monoclonal antibody.

Adult Onchocerca voluvlus and infective larvae, but not microfilariae contain an immunodominant antigen (33,000 and 21,000 Mr in females, 39,000, 33,000, and 21,000 Mr in males, 133,000 Mr in infective larvae) which is recognized by an Onchocerca-specific mAb. The component is part of the reproductive organs and muscles. 96.2% of onchocerciasis sera contained antibodies detectable by immunoblotting against it. Antigen purified by immunoaffinity chromatography was specifically recognized in immunoblots by onchocerciasis sera, but not by sera from other filarial infections. The high immunogenicity, the specificity, and the occurrence in infective larvae of this antigen indicate an immunodiagnostic potential and a possible role in the immunobiology of the parasite.

Features and outcomes of patients with grade IV renal injury.

OBJECTIVE: To evaluate the clinical features and outcomes of patients who presented with grade IV renal trauma to our urban level I trauma hospital and to further refine the absolute indications for exploration and determine the outcomes of conservative management. PATIENTS AND METHODS: In all, 77 patients with grade IV traumatic renal injuries presented to our emergency department between October 1997 and October 2006. A prospective trauma database including these patients was analysed to determine the patterns of injury, operative outcomes and complications. RESULTS: A quarter of the patients had gunshot injuries, 9% had stab injuries, and 66% had blunt traumas. In all, 36% of patients required surgical exploration to treat associated non-urological injuries. There was no or microscopic haematuria in 29% of the patients. Of the 32 patients who underwent renal exploration, 63% (20/32) underwent renorrhaphy and 37% (12/32) underwent nephrectomy. In multivariate analyses, only gunshot injury, surgery for non-urological injury, and volume of blood transfused were significantly associated with the need for renal exploration (P = 0.015, P = 0.041, and P = 0.032, respectively). The renal complication rate was higher in patients managed conservatively vs those who underwent surgical exploration, but this was not statistically significantly different (28% vs 13%, P = 0.2). Hospital stay was longer after renal exploration than after conservative management at a median of 12 days vs 7 days (P = 0.01). CONCLUSIONS: While almost all patients with penetrating injury require renal exploration, only 20% of those with blunt trauma do. Patients with no renal injuries and/or haemodynamic instability are more likely to require exploration. Finally, the rate of complications was not statistically different according to management type (conservative vs renal exploration).

Development of a highly accurate nomogram for prediction of the need for exploration in patients with renal trauma.

BACKGROUND: To develop and validate a nomogram for predicting the need for renal exploration after renal trauma. METHODS: From 1995 through 2004, 419 consecutive patients presented to our institution with traumatic renal injury. All were randomly divided into a development (50%, n = 210) and a split sample validation cohort (50%, n = 209). Logistic regression models were used to develop a nomogram for prediction of the need for renal exploration after renal trauma. Internal (200 bootstrap resamples) and 50% split sample validations were performed. RESULTS: Overall, 89 patients (21.2%) underwent renal exploration, from which 60.7% (54 of 89) underwent nephrectomy and 39.3% (35 of 89) underwent renorrhaphy. Nine percent of patients with grade II injury underwent renal exploration, 16% with grade III injuries, 41% with grade IV injuries, and 100% of grade V injuries. The kidney injury scale, the mechanism of injury, the need for transfusion, blood urea nitrogen level, and serum creatinine represented the most informative predictors and were included in the nomogram. The split sample accuracy of the nomogram for prediction of the need for renal exploration was 96.9%. It significantly (p < 0.001) exceeded the accuracy of each of its components including the American Association for the Surgery of Trauma kidney injury scale (87.7%). CONCLUSION: The nomogram generates highly accurate and reproducible predictions of the probability for renal exploration according to our decision-making. It could help standardize the management of patients with renal trauma (i.e., inclusion criteria for clinical trials) and serves as a proof-of-principle that predictive tools can be applied to the trauma setting. Its use may improve the management of renal trauma patients at institutions with limited trauma experience.

An artificial metalloenzyme with the kinetics of native enzymes.

Natural enzymes contain highly evolved active sites that lead to fast rates and high selectivities. Although artificial metalloenzymes have been developed that catalyze abiological transformations with high stereoselectivity, the activities of these artificial enzymes are much lower than those of natural enzymes. Here, we report a reconstituted artificial metalloenzyme containing an iridium porphyrin that exhibits kinetic parameters similar to those of natural enzymes. In particular, variants of the P450 enzyme CYP119 containing iridium in place of iron catalyze insertions of carbenes into C-H bonds with up to 98% enantiomeric excess, 35,000 turnovers, and 2550 hours-1 turnover frequency. This activity leads to intramolecular carbene insertions into unactivated C-H bonds and intermolecular carbene insertions into C-H bonds. These results lift the restrictions on merging chemical catalysis and biocatalysis to create highly active, productive, and selective metalloenzymes for abiological reactions.

The relationships between the burden of adult parasites, host age and the microfilarial density in human onchocerciasis.

We investigate the relationship between the microfilarial density in the skin and the burden of adult female Onchocerca volvulus by analysing pre-control nodulectomy data which allow for a direct approach, independent of exposure. The data of 169 patients in Burkina Faso and 182 patients in Liberia represent savannah and forest onchocerciasis in West Africa, respectively. Whereas in Burkina Faso, a saturating relationship between microfilarial density and worm burden suggests the operation of density-dependent processes within human hosts, the Liberian data show a linear relationship implying no density dependence. The differences may derive from differences between both parasite strains, i.e. the savannah or the forest strain of O. volvulus. Consistently for both parasite strains and independent of the worm burden, the microfilarial density increases with host age emphasising the concept of the acquisition of immunological tolerance. In male hosts in Liberia, the microfilarial density increases stronger with the worm burden than in female hosts, whereas such sex-specific differences cannot be found in Burkina Faso. In the methodological part of this investigation, we suggest the beta-distribution to be most appropriate for describing variability in microfilarial densities and we present an approach to consider the uncertainty in the adult parasite burden which cannot be determined precisely in helminth infections. Implications of density dependence are discussed with respect to immunological processes in the human host and with respect to the success of control programs. The relationships described show that regulatory processes between the parasite and the human host are multi-dimensional, operating within a high degree of biological variability.

Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus.

A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and closantel. No polymorphism could be correlated with any of the different resistances. A P-glycoprotein gene probe was also isolated from the human nematode parasite Onchocerca volvulus and an Onchocerca-specific PCR was developed.

Quantitative assessment of microfilariae and adults of Onchocerca volvulus in ethanol-fixed biopsies and nodules.

Ethanol-fixed Onchocerca nodules and skin snips were successfully digested with collagenase to assess the parasite load. The importance of this technique for investigations in the field is discussed.

Observations on adult Onchocerca volvulus maintained in vitro.

Adult Onchocerca volvulus were enzymatically isolated with collagenase from excised nodules and kept in TC medium 199 with Hank's salts supplemented with various sera. Male and female worms survived in the culture medium and 10% human serum on average for about 11 days (maximum 28 days) and 14.5 days (maximum 42 days). Up to 4,000 microfilariae were expelled by each female per day, but the production of new oocytes or development of embryos could not be observed in vitro.

Efficacy of ivermectin in the treatment of concomitant Mansonella perstans infections in onchocerciasis patients.

As part of an ivermectin dose-ranging study of onchocerciasis patients in Togo, 55 onchocerciasis patients with concomitant mansonelliasis received single oral doses either of ivermectin (100 to 200 micrograms/kg body weight) or placebo. As expected, Onchocerca volvulus microfilariae in the skin were greatly reduced in number soon after drug treatment, but microfilariae of Mansonella perstans reacted differently. Microfilarial densities of M. perstans were assessed with a filtration technique both before, and 4 times after, treatment. In untreated patients microfilarial densities were stable until the end of the study at 6 months. In patients receiving ivermectin, microfilarial densities dropped on average to less than 60% of the pre-treatment level and remained there until the final post-treatment examination. This partial reduction was probably not caused by a microfilaricidal effect of ivermectin, but rather by an altered distribution of microfilariae in the peripheral blood and in a suspected microfilarial reservoir.

Density-dependent parasite establishment suggests infection-associated immunosuppression as an important mechanism for parasite density regulation in onchocerciasis.

The modulation of human immune response by filarial parasites has yielded contradictory experimental findings and attracted much controversy. We address the unresolved question of acquisition, establishment and accumulation of Onchocerca volvulus by using a modelling approach that relates computer simulations to cross-sectional data concerning parasite burdens in 913 West African onchocerciasis patients. It is shown that the acquisition of O. volvulus is not constant with host age; instead, the analysis of age profiles of parasite burdens strongly indicate the operation of immunosuppressive processes within the human host, associated with the presence of adult parasites or microfilariae. It is suggested that these processes suppress immunity against incoming infective larvae (L3), which themselves act as an immune modulating component once they have successfully overcome the barrier of concomitant immunity. Suppression of parasite-specific immunity leads to parasite establishment rates which increase along with the parasite burden, but which hardly depend on hyperendemic annual transmission potentials. Children, still immunocompetent due to low parasite burdens, acquire 0.1-0.5 adult female parasites per year, whereas older people, immunosuppressed due to high burdens, acquire 2-4 adult female parasites per year. Differences in parasite establishment between the forest and the savannah strains of O. volvulus are quantified and dynamic aspects of density-dependent parasite establishment discussed.

A double-blind comparison of the efficacy and safety of ivermectin and diethylcarbamazine in a placebo controlled study of Senegalese patients with onchocerciasis.

Ivermectin (MK-933) has been compared with diethylcarbamazine (DEC) and placebo in a double-blind study in 30 adult male Senegalese patients with Onchocerca volvulus infection. 10 patients were randomly assigned to each treatment group. Ivermectin was administered as a single oral dose of 12 mg and DEC as 50 mg daily for two days and 100 mg twice daily for the following six days, total 1.3 g in eight days. Skin O. volvulus microfilaria densities remained near pre-study values in the placebo patients, but decreased rapidly with both active drugs to mean values about 2% of pretreatment (Day 8) and then increased slowly, reaching in 12 months about 4% of pre-treatment (ivermectin) and 18% (DEC). This difference is statistically significant. Clinical adverse reactions were recorded in four ivermectin, ten DEC and three placebo patients. One ivermectin and six DEC patients received steroid treatment for relief of these reactions. Serious adverse ocular changes were not seen in any patients, possibly because of the steroid therapy in the DEC patients. Adult O. volvulus from onchocercal nodules one and six months after treatment showed no effect of either drug on viability. Intra-uterine developing forms of the microfilariae appeared normal in all three treatment groups at the one month examination but deformed and degenerated forms were evident at six months in the ivermectin group but not in the DEC and placebo patients. Ivermectin as a single oral dose appears to be a safer and more effective microfilaricidal drug in human onchocerciasis than DEC in the standard multi-dose regimen.

Population dynamics of Onchocerca volvulus after 7 to 8 years of vector control in West Africa.

In an attempt to describe the changing population dynamics of Onchocerca volvulus during a period of vector control, nodulectomies were undertaken in 256 patients from ten villages in the Onchocerciasis Control Programme (OCP) and in 74 patients from two villages in an area with ongoing transmission. A total of 1198 nodules were excised and 4350 adult worms were isolated and examined for viability and productivity. In the OCP villages, the worm population is ageing and dying without replacement by new generations of parasites and various findings signal a breakdown of the worm population after about 12 years interruption of transmission. The sexual activity of the worms was significantly reduced. A Productivity Index was developed to measure the microfilariae production at the nodule level. The reduction in this index for the OCP villages correlates closely with the decline over the control period in the community microfilarial loads in the skin. The results show that it is not only the longevity of the parasite which will determine the duration of vector control, but that the reduced productivity of the ageing parasite population is of equal importance.

Cellular immune responses and cytokine production in BALB/c and C57BL/6 mice during the acute phase of Angiostrongylus costaricensis infection.

In our experimental study we were able to show that the contrasting outcome of Angiostrongylus costaricensis infection in C57BL/6 and BALB/c mice, in respect of morbidity and mortality, can be explained by divergent cellular immune responses and a different cytokine pattern in each strain. In BALB/c mice (i.e. those with high mortality), the initial high proliferation of ConA or LPS stimulated spleen cells dropped to very low levels after 2 weeks post-infection (p.i.), whereas in C57BL/6 mice (i.e. those with low mortality), only a minor reduction in lymphoproliferative responses after mitogenic stimulation was observed. The specific proliferation of spleen cells after stimulation with A. costaricensis adult worm antigen remained low in BALB/c mice throughout the experiment, but showed an augmented proliferation in C57BL/6 mice, especially from 2 weeks p.i. onwards. The mitogen-induced production of Th2-type cytokines (IL-4, IL-5, IL-6, IL-10) in spleen cell cultures remained low in BALB/c mice until 4 weeks p.i., but production of Th1-type cytokines (IL-2, IFN-gamma) was highly elevated at 14 and 28 days p.i. In C57BL/6 mice, an upregulated and balanced production of both Th1- and Th2-type cytokines was measured during the course of infection. In summary, a polarization of the immune response towards cellular hyporesponsiveness and a predominantly Th1 cytokine profile was observed in A. costaricensis infected BALB/c mice, which may contribute to pathogenesis and increased morbidity.

Ocular findings in a double-blind study of ivermectin versus diethylcarbamazine versus placebo in the treatment of onchocerciasis.

The effect of ivermectin, a new microfilaricide, was assessed in a double blind trial against diethylcarbamazine citrate (DEC) and placebo. Fifty-nine adult males with moderate to heavy infection with Onchocerca volvulus and with eye involvement were recruited from an area under Onchocerciasis Control Programme (OCP) vector control in Northern Ghana. They were randomly assigned to an eight-day treatment with ivermectin as a single dose of 12 mg on day 1 followed by placebo for the remaining seven days, or DEC, total dose 1.3 g, or placebo, and ophthalmological review was undertaken over a period of one year. DEC acted quickly to eliminate microfilariae from the eye and was associated with reactive ocular changes and in a few cases functional deficit. Ivermectin eliminated microfilariae slowly from the anterior chamber of the eye over a period of six months. The ocular inflammatory reaction was minimal and no functional deficit occurred. It is postulated that the observed slow action of ivermectin on the eye may be attributed in part to its instability to cross the blood-aqueous humour barrier because of its molecular size as a macrocyclic lactone causing microfilariae to leave the eye gradually along a newly created gradient. Ivermectin is an effective microfilaricide with minimal ocular adverse effect and could therefore be suitable for widespread application without strict supervision.

Optical attenuation characteristics of breast tissues at visible and near-infrared wavelengths.

Optical experiments are described for measuring the attenuation characteristics of breast tissues at visible and near-infrared wavelengths. Total attenuation coefficients post mortem were measured directly in thin tissue sections. They are usually within the range from 10 to 30 mm-1, are rather higher in fat than in fibroglandular specimens and decrease with increasing wavelength. The scattering phase function is strongly forward-peaked with the mean cosine of scattering in the range from 0.85 to 0.97 and appearing more forward-peaked in fat than in fibroglandular tissue. The reduced scattering coefficient is of the order of 1 mm-1 in all tissues. Absorption coefficients were measured indirectly in optically thick sections. They are typically between 0.1 and 0.5 mm-1 at wavelengths around 580 nm and an order of magnitude lower at 850 nm. At 580 nm and shorter wavelengths the absorption in carcinoma is significantly higher than in adjacent uninvolved tissue. Significant differences were observed in the first-order derivatives of the transmission spectra of carcinoma and surrounding tissues at certain infrared wavelengths. Transmission spectra measured in vivo across the wavelength range from 500 to 860 nm have a similar form to the spectra of excised samples. Linear absorption coefficients are generally of the same order of magnitude as those found in vitro although they are lower at green wavelengths.

Monte Carlo modelling of light propagation in breast tissue.

Light transport in three-dimensional plane-parallel tissue slabs has been modelled by Monte Carlo analogue simulation. The model design has allowed the study of transmission properties that are pertinent to imaging systems for the detection of breast cancer. An important aspect of the investigations is that they make use of data obtained from quantitative measurements of light scattering and absorption in normal and pathological breast tissues. It is shown that an imaging technique which used a raster scanning laser and detector arrangement and plane-parallel compression of the breast could have considerable advantages in terms of improved transmittance, spatial unsharpness and contrast. Time-of-flight gating of images is also found to be beneficial provided that the light intensities after temporal filtering remain adequate.

The accuracy of digital subtraction angiography for the quantification of atherosclerosis.

Digital subtraction angiography (DSA) allows the degree of arterial patency or stenosis to be rapidly quantified. We have assessed the accuracy with which a single-plane DSA system is able to quantify area patency by densitometric and geometric methods. Arterial phantoms were designed to test for systematic error; intra-arterial DSA images of critical lesions of the carotid bifurcation and the lower abdominal and peripheral vessels were used to determine intra- and interobserver reproducibility. The densitometric method, which was more accurate than the geometric method, had a mean systematic error of up to 4% and a mean intra-observer variability of about 15% (coefficient of variation). We have identified the principal sources of inaccuracy and ways in which it may be reduced.

New scans from old: digital reformatting of ultrasound images.

Two-dimensional images obtained using ultrasound have been digitized from videotape recordings and stored within a maximum of 240 digital memory planes to form a three-dimensional data set using a commercially available image processing unit. This data set has been manipulated to produce images in planes perpendicular to the original scan set. The reformatted images represent not only the scans that could have been obtained by rotating the scan head but also demonstrate planes that are not accessible by conventional scanning. The system has been evaluated with a tissue-equivalent phantom to determine the geometric accuracy of the reformatting process. Clinical material has also been used to evaluate the practical value of such a technique and to highlight difficulties that may be encountered in its routine use.

The development of a system for transillumination computed tomography.

A transillumination computed tomography system has been developed for studies of the breast, testes, neonatal skull and peripheral limbs. Initial experiments on test objects have emphasised the problems of scattering and geometrical dependence of lesion detection that are likely to be encountered in conventional diaphanography, but have demonstrated the feasibility of computed tomography as a means of improving the sensitivity of the transillumination technique. The problems of transillumination computed tomography have been addressed and further areas of research identified.

Ingestion of microfilariae from different sources by Culicoides nubeculosus (Diptera: Ceratopogonidae) through an artificial membrane.

Laboratory-hatched Culicoides nubeculosus midges were membrane-fed on cattle blood containing various Onchocerca microfilariae which had been isolated from the hides of red deer (Cervus elaphus) and from Australian and native German cattle. All microfilariae were ingested through the membrane except those of O. tarsicola, which were too large. Onchocerca gibsoni and O. lienalis reached the thoracic musculature, but no further development occurred. Microfilariae of O. flexuosa developed to the "sausage" stage. Microfilariae of O. gutturosa developed to the infective larval stage within 9 days at a temperature of 25 degrees C.