Amelioration of some immunological disorders caused by the faeces of the dominant true house dust mites in El-Minia Governorate, Egypt

BACKGROUND: House dust mites (HDMs) faeces are the main factor involved in respiratory disorder. The true HDMs,Dermatophagoides pteronyssinus and D. farinae, detected in the samples collected from the house dust are the most important causes of allergic disorders such as asthma. OBJECTIVE: The aim of this investigation was to study the curcuma and karkade amelioration of the allergenic immunological disorder, especially some cytokines, IgE and ROS, caused by the faeces of the dominant true HDM, D. pteronyssinus and D. farinae in valley and desert houses in EL-Minia Governorate, respectively. METHODS: HDM cultures, faeces isolation, plant extraction and ELISA techniques were used. Male albino rats were classified into control, inhaled, and treated groups. RESULTS: The present immunological study on the dominant allergenic true HDMs, D. pteronyssinus and D. farinae, revealed that significantly higher serum levels of TNF-α, IL-1β, IL-4, IL-13 and IgE were found in rats treated with both D. pteronyssinus and D. farinae faeces than the other groups. In addition, statistical analysis of ROS data showed significant difference between the curcuma- and karkade-treated groups and either the control or the faeces-treated groups (P < 0.05). CONCLUSIONS: Some immunological disturbances caused by repeated exposure to the faeces of two dominant allergenic true HDM species (D. pteronyssinus and D. farinae) in the valley and desert houses could be ameliorated by curcuma and karkade

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A transcription inhibitor, actinomycin D, enhances HIV-1 replication through an interleukin-6-dependent pathway.

We previously demonstrated that Actinomycin D (ActD) enhanced HIV-1 replication in the MT-2 cell, a human T-cell leukemia virus type-1-infected cell line. The MT-2 cell is known to produce multiple cytokines spontaneously. In this study, we investigated the impact of ActD on the cytokine production from MT-2 cells and HIV-1 replication in a latently infected cell line, U1. MT-2 cells were pulse-treated with 0 or 200 nM of ActD, and culture supernatants were collected 3 days after incubation. Supernatants from untreated cells (Sup0) induced HIV-1 replication by 150-fold in U1 cells. Culture supernatants from ActD-treated cells (Sup200) enhanced HIV-1 replication by 1200-fold. A combination of a sequential chromatographic approach and mass spectrometric analysis identified that the HIV-inducing factors in Sup200 were interleukin (IL)-6 and tumor necrosis factor (TNF)-beta. Quantitative analysis revealed that ActD treatment increased the concentration of IL-6 in Sup200 by 600% compared with that in Sup0 but decreased the amount of TNFbeta in Sup200 by 85%. Northern blot analysis showed that ActD treatment increased IL-6 transcripts; however, no change was seen in TNFbeta transcripts. These results suggest that ActD induces replication of HIV-1 through modulation of cytokine production.

[Expression, purification and identification of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27)].

AIM: To construct prokaryotic expression vector of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27) and express the protein in E.coli. METHODS: The rhLT-alphaDeltaN27 gene was amplified by RT-PCR using total RNA extracted from Jurkat cells,cloned into prokaryotic expression vector pET-23b, and transformed into E.coli BL21(DE3). The recombinant protein was expressed after IPTG induction and purified by DEAE Sepharose FF and Phenyl-Sepharose FF. RESULTS: The recombinant protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein. After purification, the purity of rhLT-alphaDeltaN27 was 99%, and the biological activity was more than 8x10(7) U/mg. Other characteristics of rhLT-alphaDeltaN27, such as relative molecular mass(M(r)), pI and N-terminal amino acid sequence, all corresponded to theoretical prediction. CONCLUSION: The expression vector of rhLT-alphaDeltaN27 gene was constructed, and the recombinant protein was expressed in E.coli successfully.A method of for purifying rhLT-alphaDeltaN27 was established.

Expression of lymphotoxins and their receptor-Fc fusion proteins by baculovirus.

The tumor necrosis factor (TNF) cytokine and receptor superfamily plays critical roles in immune physiology. Several members of this family, such as the lymphotoxins (LT alpha and LT beta), Fas ligand, and TNF, induce cell death in some normal and transformed cells, but also induce cell growth and differentiation. The receptors for these ligands, when expressed as fusion proteins with the Fc region of IgG, function as potent antagonists of biological activity. The receptor-Fc fusion protein is a highly versatile reagent that can be utilized in virtually all the formats designed for antibodies. In this chapter we describe the expression, purification, and assays for lymphotoxins and their receptors, using a recombinant baculovirus system.

Detection of mRNAs in Peyer's patches of the developing mouse embryo.

We describe a method to identify cells expressing mRNA of interest in the developing digestive tract by whole mount in situ hybridization with digoxigenin-labeled RNA probes. In preparing samples, serosal tissue surrounding the intestine was removed. Enzymatic reactions and probe concentrations were optimized. Furthermore, polyvinyl alcohol was included in the reaction mixture for the color development of alkaline phosphatase conjugated to the antibody against digoxigenin. These modifications improved the sensitivity and enabled us to identity cells that express mRNA in embryonic intestine. Using the antisense probe for VCAM-1, the protein product of which is an immunohistochemical marker of the Peyer's patch in the embryonic intestine, cells expressing mRNA were identified as spot-like clusters in Peyer's patches, confirming the validity of the method. With this method, mRNAs of both lymphotoxins alpha and beta, key molecules for peripheral lymphoid organ development, were found to be confined to the Peyer's patch in the developing intestine. Whole mount in situ hybridization analysis is a useful tool for exploring spatio-temporal expression profiles of mRNA in the developing immune organs.

[Preparation of lymphotoxin and properties of it]. / Poluchenie limfotoksina i izuchenie ego svoistv.

The process of isolation of highly purified human lymphotoxin was studied and optimized. A set of methods providing an increase in the content of the target product in the biomass (plasmid DNA amplification and selection of clones of transformed cells) was applied at the stage of cultivation. A two-step purification scheme (ion-exchange chromatography on DEAE-Biotone A1 and hydroxylapatite) was developed. A number of physical characteristics were studied.

Identification of two lymphotoxin beta isoforms expressed in human lymphoid cell lines and non-Hodgkin's lymphomas.

Two isoforms of lymphotoxin beta (LTbeta) were isolated from mRNAs of a panel of human lymphoid cell lines and tumor tissues obtained from patients with non-Hodgkin's lymphoma (NHL). The truncated LTbeta mRNA variant lacked 46 base pairs complementary to the complete sequence of exon 2, suggesting that both isoforms are produced by an alternative splicing mechanism. Skipping out of exon 2 causes a reading frame shift and a premature stop codon in the LTbeta mRNA variant. The predictive translated polypeptide would correspond to a severely shortened LTbeta protein that would lack the majority of the extracellular domain of the native molecule, thus impairing its normal complex assembly with LTalpha. These observations provide new insights into the molecular heterogeneity and biological function of LTbeta within the tumor necrosis factor and LT ligand-receptor system.

Role of polyethyleneimine in the purification of recombinant human tumour necrosis factor beta.

The chromatographic behaviour of recombinant human tumour necrosis factor beta (rhTNF-beta) (pI approximately 9.0) during cation-exchange chromatography at pH 7.5 is investigated. Without prior treatment of the Escherichia coli cell extract with polyethyleneimine (PEI), very little rhTNF-beta was bound to the column. However, upon addition of 5% PEI (100 microliters ml-1) to the cell lysate, rhTNF-beta was shown to bind to cation-exchange columns normally. TNF-beta was readily precipitated from the clarified cell extract by 20% ammonium sulphate, but ony ca. 25% of this precipitate could be re-solubilized for further purification. However, when 5% PEI was included in the solubilization buffer, the balance of the rhTNF-beta could be recovered. It is proposed that charge interaction between rhTNF-beta and nucleic acids in the cell extract is responsible for both of these anomalous phenomena, and that PEI (a cationic polyelectrolyte) was able to disrupt this interaction by displacing rhTNF-beta from the charge complex.

[Recombinant human lymphotoxin: synthesis in Escherichia coli cells, purification, and certain properties]. / Rekombinantnyi limfotoksin cheloveka: sintez v kletkakh Escherichia coli, ochistka i nekotorye svoistva.

The biosynthesis of recombinant human lymphotoxin produced by E. coli SG20050/pLT21 cells and deprived of 21 amino acid residues has been studied. It has been shown that the bulk of the recombinant protein in E. coli cells is in the soluble form and predominantly localized in the cytoplasm. The maximal synthesis of soluble recombinant lymphotoxin is achieved during 24-hour cultivation of producing strain cells at 32 degrees C. A procedure for isolation and purification of the recombinant protein from E. coli cells has been developed. The purification is accomplished by gel-filtration on Sephadex G-150, ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex, resulting in 97-fold purification and a 62% yield. The specific activity of the protein is about 1-10(8) U per mg of protein. Some physico-chemical properties of the recombinant protein have been studied.

Antibody against branched epitope as an affinity ligand to separate the parent protein.

A peptide that contained one of the continuous epitopes of recombinant human lymphotoxin (rhLT) (amino acid residues 139-154) has been located by epitope mapping. The branched form of this peptide was synthesized by the multiple antigen peptide procedure with an octameric branched resin and was subsequently used to elicit anti-epitope antibody in rabbits. The resulting anti-epitope was then used as an immunoaffinity ligand in affinity chromatography to purify the parent protein, rhLT, from the host cell lysate directly. It is suggested that this approach would be a general way to create novel biospecific ligands for affinity separations.

Rapid purification of recombinant human tumor necrosis factor beta.

A rapid and improved method for the purification of recombinant human tumor necrosis factor beta (rhTNF-beta) from Escherichia coli HB 101 cells has been developed. The method utilized sequential steps of polyethylenimine (PEI) and ammonium sulfate precipitation to remove most of the extraneous proteins and nucleic acids from the cell extracts. The final step of purification consisted of DEAE-Sepharose chromatography at pH 7.5 in which rhTNF-beta was eluted with starting buffer. This procedure, when compared to the earlier methods of purification, is highly efficient since we could increase the overall yield of rhTNF-beta and reduce the purification time considerably. The final yield that we obtained from 1 liter of fermentation broth (containing approximately 80 g of wet cells) was 40-50 mg.

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies.

High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.

Attempts to isolation and characterization of autolymphocytotoxins from syphilitic sera.

To solve problem if autolymphocytotoxins (AL) present in syphilitic sera are not circulating immune complexes (CIC), sera containing strong AL activity and weak CIC were precipitated with polyethylene glycol (PEG). Received PEG-precipitates were filtrated on Bio-Gel A-1.5 column to separate CIC from AL. The Al were found in the first three after void volume fractions of the column. The fractions were examined on content of protein, sugar and sialic acid. The only correlation seen refers to the sugar and protein. The fraction which display the AL activity have the ratios of sugar to protein higher than non active fractions. The data indicated besides that AL is not CIC because it is not immunoglobulin nor Treponema pallidum antigen. For further biochemical characterization of these factors more material is needed.

Production & purification of recombinant tumour necrosis factor-beta.

The effects of medium composition, temperature and tryptophan concentration on the growth and expression of a recombinant E. coli producing human tumour necrosis factor-beta (TNF-beta) were examined in shake flask cultures. We found that lower cultivation temperatures of 25 degrees C and 30 degrees C gave the best yield of soluble TNF-beta. A higher expression of total TNF-beta was obtained in defined medium. Fed-batch fermentations further confirmed that a lower mu was critical to obtaining high TNF-beta expression. This was shown to be due to the dilution effect at high mu, which affected the cell plasmid content. We found that we were unable to repress TNF-beta expression with tryptophan and TNF-beta was expressed in non-induced cultures. This has been attributed to the nature of the constructed clone, which is a low aporepressor producer, but carried a high copy number plasmid with a mutated rom gene. A rapid and improved method for the purification of TNF-beta has also been developed. The method utilised sequential steps of polyethyleneimine (PEI) and ammonium sulphate precipitation to remove most of the extraneous proteins and nucleic acids from the cell extracts. This was followed by DEAE chromatography. This procedure was found to be highly efficient and was used to purify large quantities of TNF-beta. Compared to an earlier protocol which did not include the PEI step, yields were higher and processing time was much shorter.

Natural human tumor necrosis factor beta (lymphotoxin). Variable O-glycosylation at Thr7, proteolytic processing, and allelic variation.

Natural human tumor necrosis factor beta (TNF-beta) purified from supernatants of a human B-lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N-glycosylated at Asn62; N-glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O-glycosylated at Thr7; O-glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full-length protein by trypsin-like proteolytic cleavage in the N-proximal region; these clipped molecules lack O-linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.

Recombinant human tumor necrosis factor-beta entrapped in liposomes formed by a modification of the dehydration-rehydration method retains potent cytotoxic activity on L929 cells in vitro.

Recombinant human tumor necrosis factor-beta (rhTNF-beta) may be encapsulated with high efficiency in phosphatidylcholine and distearoylphosphatidylcholine liposomes, with entrapment values of 93.4% and 92.3%, respectively, by first entrapping the substance in multilamellar vesicles using a high solute-to-phospholipid ratio followed by freeze-drying and then rehydration. The entrapped cytokine retains potent cytotoxic activity on L929 cells in vitro, causing 100% cytotoxicity, equal to that of free rhTNF-beta at a concentration of about 5 x 10(-8) g/ml.

Studies of membrane-associated and soluble (secreted) lymphotoxin in human lymphokine-activated T-killer cells in vitro.

The expression, release, and cytolytic activity of membrane-associated lymphotoxin was examined in cultures of phytohemagglutinin-P (PHA-P) and interleukin 2 (IL-2)-stimulated human T-lymphokine-activated killer (T-LAK) cells in vitro. Lymphotoxin (LT/TNF-beta) was identified on the membrane of T-LAK cells using flow cytometry. The membrane form of LT (mLT) is detected not only on T-LAK cells but also on LT-secreting human B-cell lymphoid cell lines, RPMI 1788 and Raji, but not on U937 or K562 cells. Maximum expression of mLT on T-LAK cells and the secretion of LT into the supernatant depended on the concentration of IL-2. Expression of mLT on T-LAK cells was reduced by stimulation with PHA-P; however, supernatant LT levels greatly increased. Both expression of mLT and release of soluble LT was reduced after incubation of T-LAK cells with actinomycin D (ActD) or cycloheximide (CHx). Paraformaldehyde-fixed T-LAK cells caused cytolysis of WEHI 164 cells in vitro, which was blocked by anti-LT but not anti-TNF antibody. These data support the concept that mLT may be an intermediate form to secreted LT, and that the mLT form is cytolytically active.