Functional responses and costimulator dependence of memory CD4+ T cells

To examine the functional characteristics of memory CD4+ T cells, we used an adoptive transfer system to generate a stable population of Ag-specific memory cells in vivo and compared their responses to Ag with those of a similar population of Ag-specific naive cells. Memory cells localized to the spleen and lymph nodes of mice and exhibited extremely rapid recall responses to Ag in vivo, leaving the spleen within 3-5 days of Ag encounter. Unlike their naive counterparts, memory cells produced effector cytokines (IFN-gamma, IL-4, IL-5) within 12-24 h of Ag exposure and did not require multiple cycles of cell division to do so. Memory cells proliferated at lower Ag concentrations than did naive cells, were less dependent on costimulation by B7 molecules, and independent of costimulation by CD40. Furthermore, effector cytokine production by memory cells also occurred in the absence of either B7 or CD40 costimulation. Lastly, memory cells were resistant to tolerance induction. Together, these findings suggest that the threshold for activation of memory CD4+ cells is lower than that of naive cells. This would permit memory cells to rapidly express their effector functions in vivo earlier in the course of a secondary immune response, when the levels of Ag and the availability of costimulation may be relatively low.     

Chemokine monokine induced by IFN-gamma/CXC chemokine ligand 9 stimulates T lymphocyte proliferation and effector cytokine production

Monokine induced by IFN-gamma (MIG; CXC chemokine ligand (CXCL)9) is important in T lymphocyte recruitment in organ transplantation. However, it is not known whether this chemokine, in addition to its chemotactic properties, exerts any effect on T lymphocyte effector functions. For in vivo studies, we used a previously characterized murine model of chronic rejection. The recipient mice were treated with anti-MIG/CXCL9 Ab; graft-infiltrating cells were analyzed for IFN-gamma production. For in vitro studies, exogenous CXCR3 ligands were added to CD4 lymphocytes in MLRs, and the proliferative responses were measured. Separate experiments quantitated the number of IFN-gamma-producing cells in MLRs by ELISPOT. Neutralization of MIG/CXCL9, in the in vivo model, resulted in significant reduction in the percentage of IFN-gamma-producing graft-infiltrating T lymphocytes. In vitro experiments demonstrated that 1) exogenous MIG/CXCL9 stimulated CD4 lymphocyte proliferation in a MHC class II-mismatched MLR, 2) MIG/CXCL9 also increased the number of IFN-gamma-producing CD4 lymphocytes in ELISPOT, 3) neutralization of MIG/CXCL9 in MLR reduced T lymphocyte proliferation, 4) IFN-gamma-inducible protein 10/CXCL10 and IFN-inducible T cell alpha chemoattractant/CXCL11 had similar effects on T lymphocyte proliferation, 5) MIG/CXCL9 stimulated T lymphocyte proliferation in MHC class I- and total MHC-mismatched MLRs, 6) neutralization of CXCR3 reduced MIG/CXCL9-induced T lymphocyte proliferation and the number of IFN-gamma-positive spots on ELISPOT, and 7) the proliferative effects of MIG/CXCL9 were mediated via an IL-2-independent pathway and were controlled by IFN-gamma. This study demonstrates that MIG/CXCL9 stimulates T lymphocyte proliferation and effector cytokine production, in addition to its chemotactic effects. This novel observation expands our current understanding of MIG/CXCL9 biology beyond that of mediating T cell trafficking.     

Assessment of DHPLC usefulness in the genotyping of GSTP1 exon 5 SNP: comparison to the PCR-RFLP method

Single-nucleotide polymorphism (SNP) analysis can be performed by several methods such as PCR-RFLP, real time PCR and mass spectrometry. Denaturating High Pressure Liquid Chromatography (DHPLC) analysis allows the detection of DNA mutations in heteroduplex samples. GSTP1 exon 5 gene presents a single-nucleotide polymorphism (a to g) that results into an amino-acid substitution (Ile to Val). Ile and Val variants are identified respectively by a and b alleles. This polymorphism affects enzyme activity and is highly frequent within Caucasian populations and therefore widely studied in the context of SNP related to cancer susceptibility. Our goal was to evaluate DHPLC usefulness in detecting a well-known SNP in comparison to PCR-RFLP, in the field of molecular epidemiological studies. Fifty Caucasian people were genotyped by both methods. Heterozygous samples were identified easily at two temperatures using the DHPLC method. Discrimination between a/a and b/b homozygous genotypes was done by pooling every homozygous sample with a known a/a sample. Our genotyping using both methods resulted in the characterisation of 32 (64%) a/a homozygous, 18 (36%) a/b heterozygous and 5 (10%) b/b homozygous. All samples were also identically genotyped by the two methods. Our results show that DHPLC is a good alternative to classical PCR-RFLP method in genotyping SNPs. Advantages of this chromatographic method were no restriction site needed and a reduced technical time thanks to an automated injection. Moreover, unlike classical RFLP gel analysis, DHPLC chromatograms provided objective criteria for sample classification.     

Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.     

Prevalence of congenital hypothyroidism in Isfahan, Iran: results of a survey on 20,000 neonates

AIMS: To evaluate the prevalence of congenital hypothyroidism (CH) in a screening program performed for the first time in Isfahan, Iran. METHODS: From May 2002 to December 2002, T4 and TSH serum concentrations of 20,000 3- to 7-day-old newborns, born in all 17 hospitals of the city, were measured by radioimmunoassay and immunoradiometric assay, respectively. The newborns with abnormal screening results (TSH >20 mIU/l, T4 <6.5 microg/dl and based on the weight) were re-examined. RESULTS: Of 531 recalled subjects (recall rate 2.6%), 54 were confirmed to be hypothyroid, showing a prevalence of 1:370 for CH. CONCLUSION: Considering the high frequency of CH, the necessity of implementing a routine screening program in the healthcare system of Isfahan Province is emphasized.     

The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis

Activation of NF-kappaB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IkappaB kinase (IKK) complex that phosphorylates IkappaB and leads to its dissociation from the NF-kappaB complex, thus permitting activation of NF-kappaB. The IKK complex contains primarily IKKalpha, IKKbeta, and the regulatory kinase IKKgamma, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKalpha and IKKbeta, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-kappaB. Given the pivotal role of cytokine-induced NF-kappaB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-kappaB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-kappaB in osteoinflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis.     

Molecular cloning of an alpha-enolase from the human filarial parasite Onchocerca volvulus that binds human plasminogen

Enolase represents a multifunctional protein involved in basic energy metabolism and plasminogen binding and activation at the surface of prokaryotic pathogens. A complete cDNA of 1615 bp of an alpha-enolase from Onchocerca volvulus (Ov-ENO) was isolated using a PCR-based approach. The open reading frame encoded for 435 amino acids and the high degree of conservation included the crucial amino acid residues that participate in the formation of the catalytic site, Mg(2+) binding site, and a hydrophobic motif reported to relate to surface expression. A 1089-bp fragment was expressed in a N-terminal 6 x His-tag expression vector in Escherichia coli. By immunohistological analysis using anti-Ov-ENO rabbit antibodies, native enolase could be detected in most tissues of adult O. volvulus, microfilariae, and infective larvae. Intense staining was observed in the muscles, where the energy consumption is high. The purified recombinant protein fragment revealed plasminogen binding activity in a blot-overlay assay employing anti-plasminogen antibodies. In sera from individuals infected with O. volvulus, IgG antibodies reactive with recombinant Ov-ENO were demonstrated by immunoblot and enzyme-linked immunosorbent analyses. The plasminogen-binding property of O. volvulus alpha-enolase may support plasmin-mediated proteolysis including degradation of host's extracellular matrix thereby promoting the migration of larval stages through tissues. The recognition by antibodies in sera of O. volvulus-infected persons indicate an involvement of the protein in the interaction between the parasite and the human host.     

Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress

Proline is an important component of salt-stress responses of plants. In this study the role of proline as part of salt-stress signalling in the desert plant Pancratium maritimum L. was examined. The data showed that salt-stress brought about a reduction of the growth and protein content, particularly at 300 mM NaCl, that was significantly increased by exogenous proline. In the leaves, salt-stress up-regulated ubiquitin, a small protein targeting damaged proteins for degradation via the proteasome, up to 5-fold as detected by western blotting. This change was also affected by proline even in non-stressed leaves. However, salt-stress resulted in a decrease in the amount of ubiquitin-conjugates, particularly in the roots, and this effect was reversed by exogenous proline. Severe salt-stress resulted in an inhibition of the antioxidative enzymes catalase and peroxidase as revealed by spectrophotometric assays and activity gels, but the activity of these enzymes was also maintained significantly higher in the presence of proline. Salt-stress also up-regulated several dehydrin proteins, analysed by western blotting, even in non-stressed plants. It is concluded that proline improves the salt-tolerance of Pancratium maritimum L. by protecting the protein turnover machinery against stress-damage and up-regulating stress protective proteins.     

Relationship between intrafollicular concentrations of parathyroid hormone-related peptide (PTHrP) and steroid hormones in oestrogenic and non-oestrogenic ovarian follicles in the mare

The objective of the present study was to determine whether parathyroid hormone-related peptide (PTHrP) is present in the equine follicular fluid and if so, how it is related to the follicular development in the horse. For this purpose, ovaries were collected from 40 Thoroughbred and Thoroughbred Cross mares at slaughter during the period from February to May. Normal growing follicles were dissected from the ovaries of each mare and their diameters measured. A total of 174 follicles was used in this study. The follicular fluid was aspirated from each follicle and assayed for PTHrP, oestradiol (E), testosterone (T) and progesterone (P). The follicles were classified as either oestrogenic or non-oestrogenic if the follicular fluid content of oestradiol was >40 or <40 ng/ml, respectively. PTHrP concentrations were significantly (P<0.05) higher in oestrogenic follicles, but T and P concentrations did not differ. Furthermore, E:T ratio was significantly (P<0.05) greater in oestrogenic follicles compared to the non-oestrogenic ones. The mean diameter of oestrogenic follicles was significantly (P<0.05) greater than that of non-oestrogenic ones. The higher concentrations of PTHrP observed in the follicular fluid of healthy oestrogenic follicles suggest that it may have a role in the control of ovarian function.