Cloning of the Repertoire of Individual Plasmodium falciparum var Genes Using Transformation Associated Recombination (TAR)

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member

Effect of IVIG Formulation on IgG Binding to Self- and Exo- Antigens In Vitro and In Vivo.

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid β (Aβ) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aβ. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aβ, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aβ and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aβ and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aβ or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data

Characterization of the small exported Plasmodium falciparum membrane protein SEMP1.

Survival and virulence of the human malaria parasite Plasmodium falciparum during the blood stage of infection critically depend on extensive host cell refurbishments mediated through export of numerous parasite proteins into the host cell. The parasite-derived membranous structures called Maurer's clefts (MC) play an important role in protein trafficking from the parasite to the red blood cell membrane. However, their specific function has yet to be determined. We identified and characterized a new MC membrane protein, termed small exported membrane protein 1 (SEMP1). Upon invasion it is exported into the RBC cytosol where it inserts into the MCs before it is partly translocated to the RBC membrane. Using conventional and conditional loss-of-function approaches we showed that SEMP1 is not essential for parasite survival, gametocytogenesis, or PfEMP1 export under culture conditions. Co-IP experiments identified several potential interaction partners, including REX1 and other membrane-associated proteins that were confirmed to co-localize with SEMP1 at MCs. Transcriptome analysis further showed that expression of a number of exported parasite proteins was up-regulated in SEMP1-depleted parasites. By using Co-IP and transcriptome analysis for functional characterization of an exported parasite protein we provide a new starting point for further detailed dissection and characterisation of MC-associated protein complexes

Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C-terminal NPY analogues

1. Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors. 2. In this study, the histamine releasing ability of neuropeptide Y (NPY), two C-terminal segments of NPY and 13 centrally truncated NPY analogues was examined. These results were compared with the ability of the peptides to bind to the Y2 receptor in the rabbit kidney membrane model and with their hypotensive actions in the anaesthetized-rat model. 3. All analogues tested, with the exception of [Glu4,25,33,35]-NPY(1–4)-Ahx-(25–36) and [Asp4,25,33,35]NPY(1–4)-Ahx-(25–36) which were devoid of histamine releasing activity, evoked a dose-dependent histamine release but there were marked differences between the peptides. The native peptide was the least active. 4. Histamine release was not linked to the ability of the peptides to displace NPY from Y2 receptors. There was a statistical correlation between the hypotensive effects expressed as ED10 values (μmol kg−1, which induced a blood pressure decrease of 10 mmHg) and the EC25 for histamine release (r = 0.62, P = 0.04), although histamine release may not be the sole determinant of the alterations in blood pressure. 5. There was a strong negative correlation between EC25 for histamine release and net positive charge (r = −0.93, P = 5.7 × 10−7), i.e. increasing the net positive charge caused greater histamine release. However, there was a 12 fold difference in activity amongst the most positively charged analogues (+ 5). Helicity did not correlate with histamine releasing ability. 6. In the development of NPY-related drugs the avoidance of compounds with net positive charge is recommended

<i>In vitro</i> Comparison of reformulated IgG preparations activity to Aβ, actin and tetanus toxoid by ELISA and Octet.

<p>Unformulated Privigen (in H₂O) was formulated in Proline pH 4.8, Glycine pH 4.7 or Glycine pH 4.2. (A) ELISA measurements on plate-bound Aβ oligomers. The panel shows combined results from repeated measurements (n = 3). (B) Octet binding measurements to immobilized Aβ and <b>C</b> to bound tetanus toxoid. Privigen Fc fragments were used as negative controls.</p

<i>In vivo</i> comparison of the binding to Aβ, Actin, tetanus toxoid and Varicella Zoster Virus by ELISA.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, respectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and the binding capacity to oligomeric Aβ (A and B), actin (C and D), Tetanus Toxoid (E and F), and VZV (G and H) was analyzed by ELISA. (*p<0.05, **p<0.01). Each ELISA was performed 3 times n = 3 (with all animals n = 30) except VZV t = 2 min n = 2 (with all animals n = 30) due to limited sample volume.</p

<i>In vivo</i> pharmacokinetics of different IgG preparations, proline and glycine in rats.

<p>Clr:CD(SD) rats (n = 10 in groups 0 min and 2 min; n = 5 in groups 6h and 24h) were intravenously injected with 500 mg/kg of Privigen 10%, Gammagard 10%, Octagam 10% or Gamunex 10%, repectively. Blood was taken at baseline (0 min) as well as 2 min, 6h and 24h after injection. Plasma (10% citrate) was prepared and analyzed for total IgG by Nephelometry and Proline and Glycine concentration by HPLC. The elimination profile for IgG is shown in (A) and for proline and glycine in (B).</p

Transcriptional changes in absence of SEMP1 (microarray data).

<p>Transcriptional changes in absence of SEMP1 (microarray data).</p