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PubMed2005

Analysis of putative RNase P RNA from orthopoxviruses.

A putative RNase P RNA gene in camelpox virus, one of the orthopoxviruses, was cloned and transcribed in vitro. No RNase P activity could be detected in vitro from camelpox virus RNase P RNA alone, or by addition of the Escherichia coli RNase P protein subunit to reaction mixtures. Camelpox virus RNase P RNA reconstituted in vitro with camel or HeLa cell extracts, which were pre-treated with micrococcal nuclease to degrade the endogenous RNase P RNA, showed no RNase P activity. Vaccinia virus, another orthopoxvirus, showed no RNase P activity in vaccinia-infected HeLa cells, even though transcription of the vaccinia RNase P RNA could be identified in the cells by both Northern blot and RNase protection assay. Camelpox virus RNase P RNA inhibited an endogenous HeLa RNase P activity by 20% in our assays. The 5S RNA showed no significant inhibition in this assay.

Animals↗

PubMed2024

Nanobodies: From High-Throughput Identification to Therapeutic Development.

The camelid single-domain antibody fragment, commonly referred to as a nanobody, achieves the targeting power of conventional monoclonal antibodies (mAbs) at only a fraction of their size. Isolated from camelid species (including llamas, alpacas, and camels), their small size at ∼15 kDa, low structural complexity, and high stability compared with conventional antibodies have propelled nanobody technology into the limelight of biologic development. Nanobodies are proving themselves to be a potent complement to traditional mAb therapies, showing success in the treatment of, for example, autoimmune diseases and cancer, and more recently as therapeutic options to treat infectious diseases caused by rapidly evolving biological targets such as the SARS-CoV-2 virus. This review highlights the benefits of applying a proteomic approach to identify diverse nanobody sequences against a single antigen. This proteomic approach coupled with conventional yeast/phage display methods enables the production of highly diverse repertoires of nanobodies able to bind the vast epitope landscape of an antigen, with epitope sampling surpassing that of mAbs. Additionally, we aim to highlight recent findings illuminating the structural attributes of nanobodies that make them particularly amenable to comprehensive antigen sampling and to synergistic activity-underscoring the powerful advantage of acquiring a large, diverse nanobody repertoire against a single antigen. Lastly, we highlight the efforts being made in the clinical development of nanobodies, which have great potential as powerful diagnostic reagents and treatment options, especially when targeting infectious disease agents.

Animals↗

PubMed2006

EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis.

In order to explore the genetic diversity within Echinococcus multilocularis (E. multilocularis), the cestode responsible for the alveolar echinococcosis (AE) in humans, a microsatellite, composed of (CA) and (GA) repeats and designated EmsB, was isolated and characterized in view of its nature and potential field application. PCR-amplification with specific primers exhibited a high degree of size polymorphism between E. multilocularis and Echinococcus granulosus sheep (G1) and camel (G6) strains. Fluorescent-PCR was subsequently performed on a panel of E. multilocularis isolates to assess intra-species polymorphism level. EmsB provided a multi-peak profile, characterized by tandemly repeated microsatellite sequences in the E. multilocularis genome. This "repetition of repeats" feature provided to EmsB a high discriminatory power in that eight clusters, supported by bootstrap p-values larger than 95%, could be defined among the tested E. multilocularis samples. We were able to differentiate not only the Alaskan from the European samples, but also to detect different European isolate clusters. In total, 25 genotypes were defined within 37 E. multilocularis samples. Despite its complexity, this tandem repeated multi-loci microsatellite possesses the three important features for a molecular marker, i.e. sensitivity, repetitiveness and discriminatory power. It will permit assessing the genetic polymorphism of E. multilocularis and to investigate its spatial distribution in detail.

Animals↗

PubMed2005

Information content versus relational knowledge: semantic deficits in patients with Alzheimer's disease.

Studies of semantic impairment in Alzheimer's disease (AD) have yielded conflicting results, some finding evidence of considerable deficits, others finding that semantic knowledge is relatively intact. How do we reconcile findings from picture naming tasks that seem to indicate semantic impairment in AD with results from certain sorting tasks that suggest intact semantics? To investigate the basis of the contradictory results described above, we conducted a study using two types of tasks: (1) picture naming; and (2) board sorting. The board sorting task we used is a simultaneous similarity judgment task, in which participants are asked to place more similar concepts closer together and less similar ones farther apart. We compared the performance of AD patients on these two tasks, using a number of different analyses that yield very different patterns of results. Our results indicate that whether patients show impairment or not depends on both the nature of the task and the subsequent analysis chosen. Specifically, tasks and analyses that focus on relational knowledge (e.g., dog is more related to cat than to camel) lead to different conclusions than those based on specific information about individual items. These findings suggest that the board sorting method, when coupled with multiple analyses, provides a more complete picture of the underlying semantic deficit in AD than previous studies have shown.

Adolescent↗

PubMed2005

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Animals↗

PubMed2003

Domain antibodies: proteins for therapy.

Occurring naturally in "heavy chain" immunoglobulins from camels, and now produced in fully human form, domain antibodies (dAbs) are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively). They are highly expressed in microbial cell culture, show favourable biophysical properties including solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display. dAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities.

Animals↗

PubMed2004

Evaluation of the card agglutination test (CATT/T. evansi) for detection of Trypanosoma evansi infection in water buffaloes (Bubalus bubalis) in Egypt.

A card agglutination test (CATT/T. evansi) was evaluated for detection of antibodies against Trypanosoma evansi (T. evansi) in experimentally and naturally infected buffaloes. Four calves were inoculated with a strain of T. evansi isolated from a dromedary camel. Parasitological examination of the calves revealed trypanosomes in the blood from days 4 to 9 post-inoculation (PI). General emaciation appeared from day 26 PI and aggravated until the end of the experiment (day 88 PI). Antibodies against T. evansi were detectable from day 8 PI till the end of the experiment. Parasitological examination of 200 water buffalo blood samples obtained from slaughterhouses revealed negative results. Serological examination of these animals showed that 48 (24%) water buffaloes had anti-T. evansi antibodies.

Agglutination Tests↗

PubMed2006

Hematological and biochemical changes in water buffalo calves (Bubalus bubalis) infected with Trypanosoma evansi.

Four water buffalo calves (Bubalus bubalis) were each inoculated intravenously with 10(6)T. evansi (camel isolate) and the fifth calf kept as non-infected control. The blood and sera of all calves were examined every 4 days during the first month post-inoculation (pi) and then once weekly until the end of the experiment (88 days pi). They were examined for hematological and biochemical changes, liver and kidney function tests. Hemoglobin concentration (Hb%), packed cell volume (PCV) and red blood cell count were significantly decreased. Total leucocytic count, lymphocytes and monocytes showed significant increase. Liver function tests revealed significant elevation in the activity of lactate dehydrogenase enzyme (LDH), globulin, total biliruben and indirect biliruben while alkaline phosphatase enzyme showed significant decrease. Kidney function tests revealed significant decrease of both creatinine and urea.

Animals↗

PubMed2006

Epidemiology of flea infestation of ruminants in Libya.

The results of an epidemiological and clinical study of flea infestations of farm animals in northern Libya is reported. Of 12,130 sheep examined from 124 flocks, 150 sheep were found to be infested with fleas from 50 different flocks. Likewise 23 goats from 2981 examined, and 11 calves from 1124 cattle examined were infested No fleas were recovered from camels or horses. Of 1861 fleas recovered from farm livestock, 1857 were Ctenocephalides felis strongylus and 4 were Pulex irritans. Dogs from farms and local clinics were also examined. Eight farms dogs were found to be infested with P. irritans. Of 79 infested dogs examined in veterinary clinics, 53 were found infested with P. irritans, 11 with Ctenocephalides felis felis, 12 had a mixed infestation of P. irritans and C. felis felis. Single dogs had mixed infestation of P. irritans and C. canis; C. felis felis and C. canis; and P. irritans, C. felis felis and Echidnophaga gallinacia. C. felis felis was also found on 15 infested cats. C. felis felis was never found on large farm animals despite frequently sharing their environment with dogs or cats. Likewise C. felis stongylus was never isolated from dogs or cats. This is consistent with the hypothesis that C. felis strongylus has become adapted to large farm animals, whilst C. felis felis is better adapted to dogs and cats. However, four stockmen were found infested with a total of 176 C. felis strongylus, which suggests that this subspecies is also a potential zoonosis. A significantly higher proportion of intensive farms had animals with flea infestation compared to semi-intensive farms. Fleas were not found in nomadic herds. Infested farm animals often presented with excoriation, alopecia, pruritus and hyperkeratitis particularly on the lower limbs. These signs are consistent with the generation of flea-bite hypersensitivity.

Animal Husbandry↗

PubMed2006

Study on the antioxidant status of rats experimentally infected with Trypanosoma evansi.

The antioxidant status of rats experimentally infected with Trypanosoma evansi isolated from a camel was studied using established parasitological, haematological and biochemical methods. The results indicated that infections in all rats resulted in a fulminating parasitaemia. Changes in blood parameters in T. evansi-infected rats indicated leukocytosis and a macrocytic hypochromic anaemia. A degree of anisocytosis was also observed. The activities of plasma glucose-6-phosphate dehydrogenase and glutathione peroxidase in whole blood of infected rats were significantly higher (p<0.05 and p<0.001, respectively) compared with control. No statistically significant difference was observed in the activity of superoxide dismutase in infected and control rats. Results obtained indicated that trypanosomosis caused oxidative stress and induced antioxidant enzymes.

Animals↗

PubMed1997

Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans.

Four species are presently recognised within the genus Echinococcus, namely Echinococcus granulosus, E. multilocularis, E. oligarthrus and E. vogeli, which are infective to humans. Evidence for strain diversity within the species E. granulosus, previously mainly based on morphological, biological and biochemical features, has been principally confirmed by recent genetic studies. Several molecular techniques are now available which allow the identification of E. granulosus strains. Epidemiological evidence and molecular studies indicate that the so-called sheep, cattle and cervid strains of E. granulosus are infective to humans, while the horse, camel and pig strains may be less or not infective, but this question warrants further studies. A recent study indicates that E. granulosus infecting patients in Poland shares close molecular affinity with a genotype of pig origin (G7) but exhibits some clear differences. Therefore, it may represent a previously undescribed genotype of E. granulosus, designated as G9. Phylogenetic analysis of molecular data has demonstrated the need to reappraise the taxonomic status of currently recognised strains. Clear evidence for strain variation in the other species of Echinococcus does not exist at present.

Animals↗

PubMed1997

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Adolescent↗

PubMed2000

Animal melioidosis in Australia.

Melioidosis was first diagnosed in Australia in sheep in 1949. While it has been considered endemic in tropical Australia, there have been animal outbreaks in southwest Western Australia and southern Queensland. Infection occurs in many species, with both latency and a wide range of clinical manifestations. Some species may develop melioidosis only if immunocompromised. Sheep and goats are particularly susceptible, resulting in the requirement for pasteurisation of tropical commercial goat's milk. Nine out of 43 (21%) goats had aortic lesions at autopsy and seven died from aortic aneurysm rupture. Transplacental transmission in goats has also been documented. Asymptomatic organ abscesses are common in pigs but bovine melioidosis is very rare. Camels moved north and an alpaca brought to Darwin have died from melioidosis. It also occurs in wildlife, including birds, crocodiles and kangaroos. Zoonotic transmission to humans is extremely unusual, but there are many similar epidemiological and clinical features of melioidosis in animals and humans. There have been three possible zoonotic cases in Australia. Molecular typing has found identical Burkholderia pseudomallei organisms from animals, humans and soil. The study of melioidosis in animals, especially the use of molecular genetic techniques for organism identification and typing, will continue to unravel aspects of the disease that remain unclear in humans.

Animals↗

PubMed2003

Structural modeling extends QSAR analysis of antibody-lysozyme interactions to 3D-QSAR.

This work shows that quantitative multivariate modeling is an emerging possibility for unraveling protein-protein interactions using a combination of designed mutations with sequence and structure information. Using this approach, it is possible to stereochemically determine which residue properties contribute most to the interaction. This is illustrated by results from modeling of the interaction of the wild-type and 17 single and double mutants of a camel antibody specific for lysozyme. Linear multivariate models describing association and dissociation rates as well as affinity were developed. Sequence information in the form of amino acid property scales was combined with 3D structure information (obtained using molecular mechanics calculations) in the form of coordinates of the alpha-carbons and the center of the side chains. The results show that in addition to the amino acid properties of the mutated residues 101 and 105, the dissociation rate is controlled by the side-chain coordinate of residue 105, whereas the association is determined by the coordinates of residues 99, 100, 105 (side chain), 111, and 112. The great difference between the models for association and dissociation rates illustrates that the event of molecular recognition and the property of binding stability rely on different physical processes.

Amino Acid Sequence↗

PubMed1998

Pathological studies on camelpox lesions of the respiratory system in the United Arab Emirates (UAE).

Three dromedary camels (Camelus dromedarius), which died from generalized camelpox with lesions in the respiratory system, were investigated. Histopathological lesions in the lung consisted of small, sometimes confluent foci of proliferated bronchial epithelium, necrosis and fibrosis. Orthopoxvirus cameli was demonstrated in all three cases by transmission electron microscopy and the virus was isolated from the lung and trachea on Dubca cells. It was proved by restriction enzyme analysis of the viral DNA that the isolates were identical. Immunohistochemical examination showed numerous poxvirus antigen-positive cells in the bronchial epithelia. Immunolabelled material was found in bronchial epithelial cells with hydropic degeneration and in infiltrating macrophages.

Animals↗

PubMed2003

Less impairment in face imagery than face perception in early prosopagnosia.

There have been a number of reports of preserved face imagery in prosopagnosia. We put this issue to experimental test by comparing the performance of MJH, a 34-year-old prosopagnosic since the age of 5, to controls on tasks where the participants had to judge faces of current celebrities, either in terms of overall similarity (Of Bette Midler, Hillary Clinton, and Diane Sawyer, whose face looks least like the other two?) or on individual features (Is Ronald Reagan's nose pointy?). For each task, a performance measure reflecting the degree of agreement of each participant with the average of the others (not including MJH) was calculated. On the imagery versions of these tasks, MJH was within the lower range of the controls for the agreement measure (though significantly below the mean of the controls). When the same tasks were performed from pictures, agreement among the controls markedly increased whereas MJH's performance was virtually unaffected, placing him well below the range of the controls. This pattern was also apparent with a test of facial features of emotion (Are the eyes wrinkled when someone is surprised?). On three non-face imagery tasks assessing color (What color is a football?), relative lengths of animal's tails (Is a bear's tail long in proportion to its body?), and mental size comparisons (What is bigger, a camel or a zebra?), MJH was within or close to the lower end of the normal range. As most of the celebrities became famous after the onset of MJH's prosopagnosia, our confirmation of the reports of less impaired face imagery in some prosopagnosics cannot be attributed to pre-lesion storage. We speculate that face recognition, in contrast to object recognition, relies more heavily on a representation that describes the initial spatial filter values so the metrics of the facial surface can be specified. If prosopagnosia is regarded as a form of simultanagnosia in which some of these filter values cannot be registered on any one encounter with a face, then multiple opportunities for repeated storage may partially compensate for the degraded representation on that single encounter. Imagery may allow access to this more complete representation.

Adult↗

PubMedDate not supplied

Detection of Crimean-Congo haemorrhagic fever virus antibodies in humans and imported livestock in Saudi Arabia.

A survey of Crimean-Congo haemorrhagic fever virus antibody, using the reversed passive haemagglutination inhibition test, was carried out in Saudi Arabia on sera from imported livestock at Jeddah seaport, as well as humans who had contact with imported animals on farms and in quarantine stations. Antibodies were detected in 3/354 (0.8%) humans, 88/2162 (4.1%) sheep, 14/432 (3.2%) goats and 1/182 (0.6%) cattle. Camel and horse sera were negative. The Sudanese small ruminants had the highest seropositivity rate, strongly suggesting that the virus was introduced from Sudan into Saudi Arabia. Contact with imported animals was not an apparent risk factor for virus transmission to humans.

Adult↗

PubMed2002

The mysterious immunoglobulin light chain.

The immunoglobulin light chain is mysterious from different points of view. In an evolutionary perspective there seems to be at least three major pathways but it is today impossible to say which of them is the most ancient one and which isotype belongs to which branch. It is also difficult to assess the contribution of the light chain to the binding capacity of the antibodies. It is possible that it differs from antibody to antibody but the fact that there are antibodies in camels and sharks without any light chain suggests that it is perhaps not necessary for good antigen binding.

Animals↗