Insa Bonow

Diverse Expression Patterns of Subgroups of the rif Multigene Family during Plasmodium falciparum Gametocytogenesis

Background The maturation of Plasmodium falciparum gametocytes in the human host takes several days, during which the parasites need to efficiently evade the host immune system. Like asexual stage parasites, immature gametocytes can sequester at various sites in the human body, and only mature sexual stages are found in the circulation. Although the fundamental mechanisms of gametocyte immune evasion are still largely unknown, candidate molecules that may be involved include variant antigens encoded by multigene families in the P. falciparum genome, such as the PfEMP1, STEVOR and RIFIN proteins. While expression of the former two families in sexual stages has been investigated earlier, we report here RIFIN expression during gametocytogenesis. Methodology/Principal Findings Variants of two previously characterized RIFIN subfamilies (A- and B-type RIFINs) were found to be synthesized in gametocytes. Immunofluorescence experiments showed A-type RIFINs to be accumulated in a crescent-shaped pattern of discrete punctate structures at the infected erythrocyte membrane, while members of the B-type family were associated with the parasite. Transcription analysis demonstrated the existence of diverse transcriptional regulation patterns during sexual differentiation and indicated variant-specific regulation of B-type RIFINs, in contrast to group-specific regulation for A-type RIFINs. Phylogenetic analysis of 5′-upstream regions showed that the rif–gene family falls into five defined clusters, designated rups (rif upstream) A1, A2, AB, B and C. In trophozoites and early gametocytes, rif variants of the rupsA2-type were preferentially expressed. Conclusions/Significance In this work we demonstrate the expression dynamics of the rif-gene family during sexual differentiation and present indications for subgroup specific regulation patterns. Therefore, our data provide a first foundation and point to new directions for future investigations of the potential role of RIFINs in gametocyte immune evasion.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

BackgroundPlasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.MethodsImmunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.ResultsAntigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.ConclusionThe localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites

In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.

Morphological demonstration of essential functional changes after in vitro and in vivo transition of infectiveOnchocerca volvulus to the post-infective stage

Transmission electron microscopic investigation of the morphogenesis ofOnchocerca volvulus through the third moult to the post-infective stage revealed essential alterations in various larval organs. Complete rebuilding of surface structures, the reduction of secretory granules in the glandular oesophagus, the unfolding of the intestine, an increase in the number of nerve fibres in the nerve ring and novel sensory papillae were significant findings. Transition from third- to fourth-stage larvae (L4) started as early as 48 h after transfer to vertebrate conditions in vivo in surrogate hosts and in vitro. After a resting period of about 60 h to enable a reduction in gland size and the loosening of the old cuticle and formation of the new one, the larvae started to cast the infective-stage cuticle. Young L4 exhibited a thin, monolayered cuticle and did not rebuild a glandular oesophagus. The body cavity widened, the intestine unfolded and the increased number of microvilli indicated the resumption of metabolic activity.

The ultrastructure of the anterior end of male Onchocerca volvulus : papillae, amphids, nerve ring and first indication of an excretory system in the adult filarial worm

A detailed morphological investigation of the anterior sensory organs, the nerve ring and a glomerulus-like structure in male Onchocerca volvulus was performed by means of electron microscopy. The 8 head papillae are arranged in the common 4 + 4 pattern of most filarial worms in circles around the mouth opening. The amphidial openings are found between the circles of inner and outer papillae on both sides of the mouth. Inside, several additional nerve axons are seen in the tissue of the anterior tip not related to one of the identified papillar structures. The inner and outer papillae exhibit a remarkably different fine structure, and are part of a complex system of at least 2 different receptor cell types at the anterior tip of the worm. The amphidial channel contains 8 modified cilia; accessory axons are associated with the cytoplasm of the sheath cell. The anterior nerve ring of male worms is located about 150 micrometers posterior from the outermost tip of the head region. It consists of several fibres coiled around the oesophagus. The comparison of the fine structure of the central nervous system did not show the expected morphological differences associated with the heterogeneous age distribution in the natural worm population. This was in contrast to previous findings with respect to tissues in different parts of the worm. The study also provides the first evidence that suggests the existence of an excretory organ in a filarial worm in the region of the anterior nerve ring. Paired glomerulus-like structures in the lateral chords and a canal formed by a projection of the basal zone of the cuticles were identified.

Ultrastructural observations on the nervous system and the sensory organs of the infective stage (L3) of Onchocerca volvulus (Nematoda: Filarioidea)

A detailed morphological investigation of the sensory organs and the nervous system of the third juvenile stage ofOnchocerca volvulus was performed at the ultrastructural level. A complex system of different receptor cells is found at the anterior and posterior end of this developmental stage. The eight papillae are arranged in two concentric circles consisting of two types of morphologically different receptors. Accessory nerve processes end free in the tip of the head. The paired amphids contain nine dendritic processes and accessory axons are seen in the surrounding cells. The basic structure of the amphids and of the circumoesophageal nerve ring is similar to that of other filarial nematodes. Two presumably neurosecretory cells are associated with the nerve ring. The reticular cytoplasm of these cells merges with the rough-surfaced endoplasmic reticulum of the lateral hypodermal chord. The paired phasmids at the posterior end of the developmental stages consist of single modified cilia that are embedded in an electron-dense mass. The receptor cell has access to the outside by a channel ending with a cuticular pore.

An unusual cause of elevated liver function tests in an elderly female.

An 84-years-old female patient was admitted to a hospital rom a nursing home in Hamburg (Germany) for the optimization f her insulin regimen for diabetes mellitus type 2. She further uffered from coronary heart disease, heart insufficiency NYHA II, cardiac arrhythmia, malignant melanoma, chronic obstructive ung disease, dementia and hypothyreosis. Her daily medication omprised a fixed insulin scheme, simvastatin, aspirin, clopidorel, molsidomine, digitoxin, metoprolol, enalapril, furosemide, ydrochlorothiazide, spironolactone, pantoprazole, allopurinol, evothyroxine, oxazepam, acetylcysteine, as well as inhaled foroterol. She had signs of diabetic polyneuropathia and was obese body mass index 31). Routine clinical laboratory investigations demonstrated eleated levels of c-reactive protein (CRP) of 38.1mg/l (<5.0) and iver enzymes GOT: 472U/l (10–50), GPT: 336U/l (10–50), -GT: 83U/l (<60), but a non-elevated bilirubin-level of 1.05mg/dl 0.2–1.4). Treatment with ciprofloxacin was started because of n asymptomatic urinary tract infection. Within the first three ays in hospital there was a further increase of liver enzymes GOT: 815U/l, GPT: 541U/l, -GT: 942U/l) but without clinical ymptoms. Ultrasound examination as well as computerized

Ultrastructure study of the excretory system and the genital primordium of the infective stage ofOnchocerca volvulus (Nematoda:Filarioidea)

The electron microscopic investigation of the anterior part of the infective third-stage juvenile ofOnchocerca volvulus provides first insights into the structure of the excretory system of this developmental stage of the parasite. The most anterior part of this system consists of a cell process of the syncytial excretory cells. At this height the excretory cells enclose the cuticle-lined excretory channel. The channel is in the process of elongation in the anterior-posterior direction, indicated by cell division in this region. More posteriad an ampulla-like structure is forming in the cytoplasm of the excretory cells. The inner surface of this ampulla is lined with a small number of single microvilli. In this part of the system the cytoplasm of the excretory cells is rich in Golgi bodies and endocytic vesicles. The ampulla has direct access to the exterior by the excretory duct. The excretory duct is a cuticle-lined structure surrounded by supporting fibres of an additional cell. This duct cell connects the excretory duct to the body-wall cuticle at the excretory pore. Adjacent to the region of the excretory system a cell is found that resembles a gland cell. This cell is in close contact to the ventral nerve cord. The genital primordia of the third-stage juvenile consist of several dividing cells. The female genital primordium is seen at the junction of the muscular with the glandular oesophagus and the male primordium can be found at the junction of the glandular oesophagus with the gut.

The anterior central nervous system of male Onchocerca volvulus (Nematoda:Filarioidea) as a target for chemotherapy: results of an electron microscopy study.

Abstract An electron microscopy study was performed to evaluate the feasibility of the use of the anterior nerve ring of male Onchocerca volvulus for the assessment of early drug effects. Worms were exposed to new and known compounds at reasonable concentrations of 1 μM and less for 6, 12, 18, and 36 h in an established in vitro system. The anterior end of the filariae up to a length of 1 mm was examined and the morphological findings were compared with motility and reduction of a tetrazolium salt to formazan by live but not dead worms. The nerve fibers were more susceptible to the chemotherapeutic intervention then the other tissues in the anteriormost part of the worms. The alterations depended on the duration of exposure and the chemical nature of the compounds used. Morphological changes in the nervous tissue and the inhibition of motility and formazan production corresponded well for the arsenical mel w, used as an active standard, two pyrimidinylguanidines (PD 105482 and PD 105666), and an imidazolinylhydrazone (WR 251993).

Evaluation of automated loop-mediated amplification (LAMP) for routine malaria detection in blood samples of German travelers – A cross-sectional study

BACKGROUND We assessed a commercial loop-mediated amplification (LAMP) platform for its reliability as a screening tool for malaria parasite detection. METHODS A total of 1000 blood samples from patients with suspected or confirmed malaria submitted to the German National Reference Center for Tropical Pathogens were subjected to LAMP using the Meridian illumigene Malaria platform. Results were compared with microscopy from thick and thin blood films in all cases. In case of discordant results between LAMP and microscopy (n = 60), confirmation testing was performed with real-time PCR. Persistence of circulating parasite DNA was analyzed by serial assessments of blood samples following malaria treatment. RESULTS Out of 1000 blood samples analyzed, 238 were positive for malaria parasites according to microscopy (n = 181/1000) or PCR (additional n = 57/60). LAMP demonstrated sensitivity of 98.7% (235/238), specificity of 99.6% (759/762), positive predictive value (PPV) of 98.7% (235/238) and negative predictive value (NPV) of 99.6% (759/762), respectively. For first slides of patients with malaria and for follow-up slides, sensitivity values were 99.1% (106/107) and 98.5% (129/131), respectively. CONCLUSIONS The performance of the Meridian illumigene Malaria platform is suitable for initial screening of patients suspected of clinical malaria.