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The network of molecular mediators involved in the transmigration of leukocytes to inflamed tissues has been expanding with the identification of new molecules involved in the inflammatory response. We have previously shown using a rat model that Protein Disulfide Isomerase PDIA4 (ERP72) is involved in the inflammatory response to skeletal muscle traumatic injury. In this paper, we report observations suggesting that calsequestrin 2 (CASQ2), another member of the thioredoxin/PDI family, might contribute to the inflammatory response that leads to adhesion and transmigration of leukocytes into injured skeletal muscle. Indeed, real time PCR assay showed that the expression level CASQ2 is significantly enhanced [p2+ buffering role of CASQ2 suggest that the protein contributes to the inflammatory process by providing the Ca2+ needed to activate the inflammasome and the leukocytes adhesion molecules which enhances transmigration of inflammatory cells into the injured muscle.

Leishmaniasis is an infectious disease caused by parasites of the genus Leishmania and transmitted by the bite of a sand fly. To date, most available drugs for treatment are toxic and beyond the economic means of those affected by the disease. Protein disulfide isomerase (PDI) is a chaperone protein that plays a major role in the folding of newly synthesized proteins, specifically assisting in disulfide bond formation, breakage, or rearrangement in all non-native proteins. In previous work, we demonstrated that Leishmania major PDI (LmPDI) has an essential role in pathogen virulence. Furthermore, inhibition of LmPDI further blocked parasite infection in macrophages. In this study, we utilized a computer-aided approach to design a series of LmPDI inhibitors. Fragment-based virtual screening allowed for the understanding of the inhibitors' modes of action on LmPDI active sites. The generated compounds obtained after multiple rounds of virtual screening were synthesized and significantly inhibited target LmPDI reductase activity and were shown to decrease in vitro parasite growth in human monocyte-derived macrophages. This novel cheminformatics and synthetic approach led to the identification of a new series of compounds that might be optimized into novel drugs, likely more specific and less toxic for the treatment of leishmaniasis.

Iron-refractory iron deficiency anemia (IRIDA) is considered an autosomal recessive iron deficiency anemia due to mutations in the transmembrane protease serine 6 (TMPRSS6) gene. Variations in iron parameters and a higher risk of iron deficiency have been linked to the TMPRSS6 mutations. Furthermore, human genome-wide association studies (GWAS) identified a common mutation (rs855791) linked to abnormal hematological parameters, highlighting the importance of the TMPRSS6 gene in the regulation of iron homeostasis. This is the first study to investigate TMPRSS6 gene mutation in six Saudi families of probands with iron deficiency anemia unresponsive to oral iron and partially responsive to parenteral iron administration. Each participant provided a vacutainer tube with three blood samples (2.5 ml each) and analyzed based on hematological, biochemical iron profiles, and followed by genotyping by PCR. The TMPRSS6 gene was amplified, sequenced, and analyzed in all probands and family members. Statistical analysis was done using SPSS and SHEsis software. Few functional mutations in these families were suggested (p.W73X, p.E523K and p.V736A). The proband of family 6 presented numerous hematological abnormalities upon initial consultation, including normocytic anemia accompanied by low Hb, normal MCV, low serum iron, low serum ferritin, and normal TIBC. While the p.W73X variant was only found in 2 families, the p.V736A variant was found in all examined Saudi families with IRIDA. Given the evidence outlined for these six cases, future genotype-phenotype correlation studies in a large number of IRIDA patients in Saudi Arabia may be very informative for patient management, in addition to increasing knowledge of TMPRSS6 function during development as well as factors in the regulation of TMPRSS6 and its effect on iron levels in the body.

The vascular inflammatory response involves the coordinated action of a large network of molecular mediators and culminates in the transmigration of leukocytes into the site of inflammation. Inflammatory mediators include a variety of protein families, including adhesion molecules such as integrins and members of the immunoglobulin superfamily, as well as other cytokines and chemokines. In this study, a rat model of traumatic skeletal muscle injury was used to demonstrate endoplasmic reticulum resident protein 72 (ERp72) overexpression in the early phase of the inflammatory response that follows skeletal muscle injury. Reverse transcription‑quantitative PCR, western blotting, dual‑labeling immunohistochemistry and immunofluorescence experiments confirmed that ERp72 was expressed on the endothelial cells of blood vessels present at the injured area. In addition, a cell‑based neutrophil adhesion assay indicated that a polyclonal antibody specific for ERp72 significantly reduced adhesion of neutrophils to activated human umbilical vein endothelial cells (35% reduction). These data suggested that ERp72 expression on vascular endothelial cells may play a role in skeletal muscle inflammation and could be considered as a target for the modulation of leukocyte‑endothelial cell interactions in an inflammatory setting.

Objective/background Mutations in transmembrane protease serine 6 (TMPRSS6) gene induce high hepcidin level, which causes iron-refractory iron deficiency anemia (IRIDA) by preventing duodenal iron absorption. This study aims to identify the common genetic variations of the TMPRSS6 gene that affect iron levels among Saudi female patients with iron deficiency anemia (IDA). Methods All study participants were Saudi females (12–49 years old): 32 patients with IDA, 32 patients with IRIDA, and 34 healthy individuals comprising the control group. Hematological investigations, iron profile, serum hepcidin level, and TMPRSS6 gene transcription were determined. The TMPRSS6 gene was amplified, sequenced, and analyzed among all study participants. Results The mean hepcidin and TMPRSS6 RNA transcription levels in IDA and IRIDA groups were significantly lower than those in the control group. TMPRSS6 gene sequence analysis detected 41 variants: two in the 5′ untranslated region (5′UTR), 17 in introns, and 22 in exons. Thirty-three variants were previously reported in the Single Nucleotide Polymorphism Database, and eight variants were novel; one novel variant was in 5′UTR (g.-2 T > G); five novel variants were detected in exons (p.W73X, p.D479N, p.E523K, p.L674L, and p.I799I). At the time of the sequence analysis of our samples, two variants—p.D479N and p.674L—were novel. However, these variants are present at a very low allele frequency in other populations (L674L, 0.00007761 and D479N, 0.000003980). Conclusion This is the first study to investigate the genetic variants of TMPRSS6 gene in Saudi female patients with IDA. The generated data will serve as a reference for future studies on IDA in the Arab population.

International audience; Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) during secondary immune response. Mutations in the AICDA gene are responsible for Hyper IgM 2 syndrome where both CSR and SHM or only CSR are affected. Indeed, triggering either of the two mechanisms requires the DNA deamination activity of AID. Besides, different domains of AID may be differentially involved in CSR and SHM through their interaction with specific cofactors. Herein, we studied the AID-induced SHM activity of the AID-His130Pro mutant identified in a patient with Hyper IgM 2 syndrome. AID mutagenic activity was monitored by the reversion of nonsense mutations of the EGFP gene assessed by flow cytometry. We found that the His130Pro mutation, which affects CSR, preserves AID mutagenic activity. Indeed, the His130 residue is located in a putative specific CSR region in the APOBEC-like domain, known to involve CSR specific cofactors that probably play a major role in AID physiological activities.

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc- (His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.

The beta2 integrin CD11b/CD18 (CR3) is a major adhesion receptor of neutrophils, normally utilized to fend off infections. This receptor contributes, however, to multiple forms of non-infectious inflammatory injury when dysregulated as shown in gene knock-outs and through the use of blocking monoclonal antibodies. The major ligand recognition site of CR3 has been mapped to the A-domain in the CD11b subunit (CD11bA). The recombinant form of this domain exhibits a ligand binding profile similar to that of the holoreceptor. To assess the potential anti-inflammatory activity of CD11bA as a competitive antagonist of CR3 in vivo, we assessed its effects on a developed animal model of traumatic skeletal muscle injury in the rat. Recombinant soluble rat CD11bA-domain fused to glutathione-S-transferase (GST) was administered intravenously in a single dose at 1 mg/kg to nine groups of Wistar rats, five in each group, 30 min before inducing traumatic skeletal muscle injury. Control animals received either a function-blocking anti-CD11b/CD18 monoclonal antibody (1 mg/kg), non-functional mutant forms of the CD11bA (D140GS/AGA, T209/A, D242/A), recombinant GST or buffer alone. In control animals, the wounded muscle showed oedema, erythrocyte extravasation and myonecrosis both within and outside the immediate wounded area (5-10 mm zone) and influx of neutrophils was detected 30 min post-wound, followed by a second wave 3 hr later. Wild-type CD11bA- or anti-CD11b monoclonal antibody (mAb)-treated rats showed a comparable and significant decrease in the number of infiltrating PMN (78 + 4%, n = 70 and 86 +/- 2%, n = 50, respectively) and preservation of the muscular fibres outside the immediate zone of necrosis (75 + 4%, n = 70, 84 +/- 1%, n = 50, respectively), compared to controls. These data demonstrate that CD11bA can be an effective tissue-preserving agent in acute inflammatory muscular injury.

CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia c oli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris . Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases ( n = 40) from BCG vaccinees ( n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli -expressed antigens. Overall, the trans -production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.

We herein report the production in the methylotrophic yeast Pichia pastoris of a recombinant scFv form of monoclonal antibody 107 [MAb 107]. This antibody is directed to the leukocyte adhesion molecule CR3 (CD11b/CD18) and is specific of the CD11bA domain. MAb 107 is a ligand mimic of CR3 that blocks the adhesion of leukocyte and their transendothelial migration (diapedesis), a feature that confers this MAb a potential anti inflammatory activity. Expression of scFv 107 as a myc-(His) 6 secreted fusion protein was achieved in the yeast P. pastoris using plasmid pICZαB. A spontaneous specific cleavage of the myc-His tag was consistently observed regardless of the culture conditions and the addition of different concentrations of casaminoacids in the medium. Loss of the myc-His tag did not affect the binding activity of scFv 107. This loss is due either to a specific proteolysis or to a mechanical cleavage. Optimization of the production of scFv 107 in shake flasks was carried out using an L16 array Taguchi design. A level of purified scFv-myc-His fusion protein estimated at 40 mg/L was reached at pH 6 and a temperature of 27 °C. The production of large amounts of recombinant scFv form of MAb 107 is essential for further molecular studies of the interactions between CR3 and its ligands, mainly those mediated by the CD11b A domain. It will also facilitate the investigation of its anti inflammatory activity in vivo.

We herein report the fabrication and characterisation of a novel impedimetric immunosensor based on an electrolyte–insulator–semiconductor (EIS) structure. It is a silicon nitride/aminosilane/glutaraldehyde/antibody biosensor specifically designed for the detection of antigens. This immunosensor was fabricated following the Self-Assembled Monolayers (SAMs) method, followed by glutaraldehyde cross linker and anti-rabbit IgG immobilization. The high coverage area of the silane monolayer on silicon nitride surface was estimated with impedance spectroscopy technique and the molecular structure with Fourier-transform infrared (FTIR) technique. The binding between antibody and antigen (Rabbit IgG) was monitored by measuring the resistance variation of the grafted layer. The developed immunosensor has a limit detection of 50 ng/ml of antigen.

We have typed 275 men from five populations in Algeria, Tunisia, and Egypt with a set of 119 binary markers and 15 microsatellites from the Y chromosome, and we have analyzed the results together with published data from Moroccan populations. North African Y-chromosomal diversity is geographically structured and fits the pattern expected under an isolation-by-distance model. Autocorrelation analyses reveal an east-west cline of genetic variation that extends into the Middle East and is compatible with a hypothesis of demic expansion. This expansion must have involved relatively small numbers of Y chromosomes to account for the reduction in gene diversity towards the West that accompanied the frequency increase of Y haplogroup E3b2, but gene flow must have been maintained to explain the observed pattern of isolation-by-distance. Since the estimates of the times to the most recent common ancestor (TMRCAs) of the most common haplogroups are quite recent, we suggest that the North African pattern of Y-chromosomal variation is largely of Neolithic origin. Thus, we propose that the Neolithic transition in this part of the world was accompanied by demic diffusion of Afro-Asiatic–speaking pastoralists from the Middle East.

Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways.

We have identified four different mutations causing leukocyte adhesion deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum includingde novoand recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

An experimental model to study the effects of viral inactivators on the biological properties of DNA was developed. Beta-propiolactone (betaPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that betaPL impairs these two major DNA functions are presented. The amounts of betaPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations.

Polymorphonuclear cells and monocytes (phagocytes) are a critical component of host defense against infections. However, these cells also play a significant role in host tissue damage in many noninfectious diseases, such as ischemia-reperfusion injury syndromes and rejection of transplanted organs. The leukocyte adhesion molecule family CD11/CD18 (beta 2 integrins) is critical to the function of polymorphonuclear cells and monocytes in inflammation and injury. Inherited deficiency of CD11/CD18 impairs phagocyte chemotaxis, adhesion and transmigration across endothelium, and clearance of invading microorganisms through phagocytosis and cell-mediated killing. Furthermore, murine monoclonal antibodies directed against the CD11b/CD18 (CR3) heterodimer have been shown to reduce, by 50%-80%, phagocyte-mediated ischemia-reperfusion injury in several organ systems, such as the myocardium, liver, and gastrointestinal tract and to inhibit development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice. Expression of CD11b/CD18 in a soluble and functional form might therefore be potentially useful as an anti-inflammatory agent. We have now expressed a recombinant soluble heterodimeric form of this human beta 2 integrin, normally expressed as two noncovalently associated membrane-bound subunits. The secreted receptor exhibited direct and specific binding to its ligand, iC3b, the major complement C3 opsonin, and inhibited binding of polymorphonuclear cells to recombinant interleukin 1-activated endothelium.

The leukocyte adhesion molecules CD11a/CD18, CD11b/CD18, and CD11c/CD18 (Leu-CAM) are members of the integrin receptor family and mediate crucial adhesion-dependent functions in leukocytes. The molecular basis for their deficient cell surface expression was sought in a patient suffering from severe and recurrent bacterial infections. Previous studies revealed that impaired cell surface expression of Leu-CAM is secondary to heterogeneous structural defects in the common beta subunit (CD18). Cloning and sequencing of complementary DNA encoding for CD18 in this patient revealed two mutant alleles, each representing a point mutation in the coding region of CD18 and resulting in an amino acid substitution. Each mutant allele results in impaired CD18 expression on the cell surface membrane of transfected COS M6 cells. One substitution involves an arginine residue (Arg593----cysteine) that is conserved in the highly homologous fourth cysteine-rich repeats of other mammalian integrin subfamilies. The other substitution involves a lysine residue (Lys196----threonine) located within another highly conserved region in integrins. These data identify crucial residues and regions necessary for normal cell surface expression of CD18 and possibly other integrin beta subunits and define a molecular basis for impaired cell surface expression of CD18 in this patient.

Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.

CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively.

International audience; A splice junction mutation at the exon 2 – intron 2 boundary of the carbonic anhydrase II (CA II) gene was previously shown to be the unique mutation underlying the CA II deficiency syndrome in patients of Arab descent. Fourteen Tunisian (Maghrebian) families with a history of osteopetrosis, renal tubular acidosis, mental retardation, and CA II deficiency were studied to test the hypothesis that the mutation, found in all 24 patients, derived from a common ancestor originating in the Arabic Peninsula. A filiation study permitted us to trace these families back to a common Arabic tribe that settled in the Maghreb in the tenth century, indicating a common ethnic origin for these families. Segregation of the mutation with a TaqI biallelic restriction site polymorphism upstream of the CA II gene was studied by sequence-tagged site analysis in all the family members. These studies showed cosegregation of the Taq (-) allele with the mutation in 12 families out of 14. This observation supports a founder effect to explain the common CA II deficiency allele in this population. In the remaining two families, a genomic recombination or gene conversion occurred between the TaqI restriction marker and the mutation causing the disease. The relatively high recombination frequency suggests the presence of a hot spot for recombination or gene conversion at the CA II locus.

We have investigated, in the genomic DNA of ten Tunisian patients, the presence of a splice junction mutation at the 5' end of intron 2 in the carbonic anhydrase II gene (CAII) previously described in six CAII-deficient patients presumed to be of Arab origin. All our patients were homozygous for this mutation and were mentally retarded, a characteristic feature of the phenotype of patients with an Arabic background. This mutation is found exclusively in patients with an Arabic background and thus may be confined to this ethnic group.

In this work we employ PEGylated polyethyleneimine (pPEI) o act both as an affinity ligand, to increase the selectivity of the ystem, and as a pDNA condensation agent. In this way we were ble to obtain plasmid polyplexes with a 100% yield without NA or protein contamination, after two ATPS extractions from lkaline lysates. In the first one a PEG 600—ammonium sulhate system, already described, was used and in the second one t was employed a PEG 3350—Dextran 110 system containing PEI. The isolation of polyplexes was carried out by ultrafiltraion. Although a substantial removal of Dextran and PEG was chieved the yield in polyplexes was very low (5–10%). This as due to binding of the polyplexes to the polyethersulphone embrane used. Further studies are being carried to optimize he ultrafiltration step. These results open the possibility of simultaneously purifyng and condensing the plasmid with the delivery vehicle for herapeutic applications and achieving the integration of two mportant steps on pharmaceutical plasmids production.

By isoelectrofocusing in agarose, the properdin factor allotype BfF could be split into two subtypes: BfFa with one major cathodic band and BfFb with the same cathodic band but in addition a major anodic band. By scanning, BfFaFb heterozygotes were distinguished from BfFbFb homozygotes by the stronger intensity of the anodic band in the latter. The two subtypes were segregated perfectly with HLA in 40 families and showed different association patterns with HLA markers. BfFa seemed to be linked to B35 while BfFb showed a strong linkage with all the components of the following haplotype: HLA-A29, Cw-, B44, BfFb, C4A3B1, DR7. The frequency of BfFa among BfFS heterozygotes was 41% (46/113) and that of BfFb 59% (67/113).

Compared with monoclonal antibodies of rodent origin, human monoclonal antibodies (HuMAbs) have the theoretical advantage of a better correlation with reaction patterns observed with alloantibodies, and in addition might have therapeutic application. A number of HuMAbs have been produced [3], but none to the major histocompatibility complex products have so far been described. The aim of our work was to evaluate the human-human and the human-mouse hybridoma systems for the production of alloreactive HuMAbs by performing parallel fusions between peripheral blood lymphocytes (PBLs) from women HLA-immunized by pregnancy and four 8-azaguanine-resistant mouse and human cell lines.