Autocrine secretion of interferon gamma negatively regulates homing of immature B cells.

The mechanism by which immature B cells are sequestered from encountering foreign antigens present in lymph nodes or sites of inflammation, before their final maturation in the spleen, has not been elucidated. We show here that immature B cells fail to home to the lymph nodes. These cells can actively exclude themselves from antigen-enriched sites by downregulating their integrin-mediated adhesion to the extracellular matrix protein, fibronectin. This inhibition is mediated by interferon gamma secretion. Perturbation of interferon gamma activity in vivo leads to the homing of immature B cells to the lymph nodes. This is the first example of autocrine regulation of immune cell migration to sites of foreign antigen presentation.

Cloning and characterization of the SmIMP25 integral membrane protein of the parasitic helminth Schistosoma mansoni.

The cDNA and genomic clones encoding a 25 kDa integral membrane protein, termed SmIMP25, were isolated from Schistosoma mansoni. The 2.2 kb SmIMP25 mRNA was found in all developmental stages of the parasite tested: miracidium, sporocyst, cercaria and adult worm. The SmIMP25 gene is at least 16 kb long and it is split by four introns ranging in size from 36 bp to > or = 9 kb. Excluding the introns, the gene and the cDNA show 100% sequence identity. The cDNA has an open reading frame encoding a protein 223 amino acids long. The predicted sequence reveals a distinct hydrophobic domain of 20 amino acids located 12 residues from the carboxyl-terminal end. The properties of this domain (marked hydrophobicity, size, flanking by charged residues and C-terminal location) are typical of the transmembrane segments of integral membrane proteins. The presence of three potential N-glycosylation sites is also consistent with membrane proteins that are often glycosylated at the extracellular domain. Accordingly we propose that SmIMP25 is an integral membrane protein in which residues 1-191 are extracellular, residues 192-211 comprise the hydrophobic domain that spans the membrane, and residues 212-223 are intracellular. The SmIMP25 was synthesized as a fusion protein in bacteria and antibodies were elicited in rabbits. Antibodies against SmIMP25 specifically precipitated a 25 kDa protein from cell-free products programmed by schistosome mRNA, in agreement with the size of the protein predicted from the cDNA sequence. Immunofluorescence studies showed SmIMP25 on the surface of the parasite. Surface molecules expressed at the host-parasite interface are likely to provide information on host parasite relationship and may serve as targets for protective immunity.

Cloning of the SmSPO-1 gene preferentially expressed in sporocyst during the life cycle of the parasitic helminth Schistosoma mansoni1.

Schistosomes are parasitic helminths with a complex life cycle in human and snail hosts. They express stage-specific genes that conceivably determine distinct properties of the parasite at different developmental stages. Here we report the stage-specific gene SmSPO-1, which is preferentially expressed in sporocysts residing in the snail host. The cDNA and the gene were cloned and sequenced. The cDNA, from cap site to the poly(A) addition site, is 498 bp long. It encodes a protein of 117 amino acids with a hydrophobic signal peptide of 18 residues, indicating that SmSPO-1 is a secreted or a membranal protein. In the gene the cDNA is split into four exons spread over 2.1 kb of chromosomal DNA.

Differences in DNA-sequence recognition between the DNA-binding domain fragment and the full-length molecule of the heat-shock transcription factor of schistosome.

Binding and inhibition studies reveal that the DNA-binding domain (DBD) fragment and the full-length molecule of the heat-shock transcription factor of schistosome (SmHSF) differ in DNA sequence recognition. SmHSF does not recognize the ideal HSE consensus sequence (nGAAnnTTCnnGAAn) but recognizes a variant HSE that contains nGTAn instead of nGAAn in the third pentamer. The DBD reacts efficiently with the ideal HSE sequence and with lower affinity with the variant HSE sequence. These findings suggest that elements inside and outside the DBD contribute to the DNA-binding specificity of HSF.

Grb7 expression and cellular migration in chronic lymphocytic leukemia: a comparative study of early and advanced stage disease.

Grb7, a noncatalytic intracellular adaptor protein involved in cell migration, is overexpressed in certain invasive and metastatic solid tumors. We found a highly significant difference in the level of expression of Grb7 between chronic lymphocytic leukemia (CLL) cells obtained from stage I and stage IV patients (P<0.001). Using semiquantitative RT-PCR, we detected high levels of Grb7 in 88% of stage IV patients vs only 18% in stage I patients. A corresponding increase was found in the in vitro migration of stage IV CLL cells in comparison to stage I cells. The statistically significant difference in the expression of Grb7 between stage IV and stage I patients was preserved even when tested specifically in the ZAP70-positive group (P<0.01). These findings show that Grb7 levels reflect the severity of the disease, and may be used, in conjunction with ZAP70, to predict disease progression.

Cloning and sequencing of an hsp70 gene of Schistosoma mansoni.

Schistosomes have a complex life cycle (vertebrate and molluscan hosts as well as larvae living freely in water) in which they are exposed to different environments and temperatures (20 degrees C - 37 degrees C). Since heat shock genes are activated in response to stress and during development [1], it is of interest to study the hsp70 gene family in schistosome. To approach this issue we have isolated from Schistosoma mansoni a genomic clone containing the complete coding region of hsp70 and the 5' flanking DNA with transcription regulatory elements including HSE (heat shock element) sequences.

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni.

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca2+-dependent electrophoretic mobility (increased with Ca2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.

Regulation of HSP70 gene expression during the life cycle of the parasitic helminth Schistosoma mansoni.

Analyses of RNA from different developmental stages of Schistosoma mansoni showed stage-specific expression of heat-shock protein 70 (hsp70), which is regulated by a developmental program and by stress. The developmental program, common to hsp70 and other genes (e.g. paramyosin), refers to constitutive expression in miracidia sporocyst and adult worm but not in cercariae, and to the termination of hsp70 gene transcription during sporocyst/cercaria transformation. Stress induction, specific to hsp70, refers to transient accumulation of high levels of hsp70 mRNA during cercariae/schistosomula transformation and in adult worms after heat shock (42 degrees C). Cercariae/schistosomula transformation can be visualized as a physiological stress involving shifts in temperature (23-37 degrees C) and in salt concentration (from water to isotonic medium), as well as removal of tails from cercariae to yield isolated bodies that transform into schistosomula. It was found that temperature is an important factor, but not sufficient for strong induction of the hsp70 genes of schistosomula. Tail removal is an obligatory step for full induction of the hsp70 genes of schistosomula, in response to a temperature shift from 23-37 degrees C. The hsp70 genes in cercariae and isolated tails do not respond to stimuli (salt and temperature increases) that strongly activate the genes in isolated bodies (i.e., schistosomula). We speculate that the hsp70 genes in intact cercariae are not inducible because the tails can produce inhibitory signals that diffuse to the bodies and suppress their hsp70 genes. This hypothesis is useful to explain the termination of hsp70 gene transcription during sporocyst/cercaria transformation by the inhibitory effect of the growing tail.

Different forms of the mRNA encoding the heat-shock transcription factor are expressed during the life cycle of the parasitic helminth Schistosoma mansoni.

Several cDNAs and a gene encoding the heat-shock transcription factor (HSF) of schistosome were cloned, and multiple forms of the mRNA were found at different developmental stages of the parasite. The encoded protein contained a DNA-binding domain with expected sequence identity (39-58%) to other HSF molecules, and two leucine zipper motifs (LZ123 and LZ4) involved in the oligomerization of HSF. Adult worms express three isoforms of HSF mRNA generated by alternative splicing inside the coding region that contains in-frame splice signals. Introns are not involved in the process since the deleted segments (36 bp or 45 bp) are not flanked by any intron in the gene. Structural variations generated by alternative splicing (insertion of 3 amino acids or 15 amino acids) are continual with LZ4 and added hydrophobic residues are in register with the hydrophobic heptad repeats of LZ4. Structural diversity at the C-terminus of LZ4 may affect the strength of LZ4 interaction with the oligomerization domain (LZ123) and thus modulate the DNA-binding activity of HSF. The conservation of this mechanism in mouse and schistosome may reflect evolutionary pressure to generate multiple HSF species exhibiting functional diversity and capable of responding to different stress signals and physiological signals. Adult worms express HSF mRNA of 2.5 kb, in agreement with the size of the cDNA, while cercariae (developmental stage preceding adult worm) show multiple bands in the range 2.5-3 kb. Available data indicate that the HSF mRNAs of cercariae are inactive. We propose that these mRNA species are generated by an alternative splicing that incorporates introns, which inactivate the mRNA by the insertion of termination codons and/or by shifting of the reading frame. Parasite HSF protein produced in bacteria showed DNA sequence recognition similar to that of HSF in parasite extracts, i.e. the recombinant HSF reacted better with a variant heat-shock element (HSE; one base change in the third NGAAN pentamer of the ideal HSE consensus sequence) than with the ideal HSE. The size of the HSF gene is 12 kb and it is composed of ten exons and nine introns. Excluding the introns, the gene and cDNA show 100% sequence identity. A plant HSF gene contains only a single intron, which matches with the position of intron I2 of schistosome. That the position of this intron is conserved in remote species is indicative of an important function during evolution of the HSF gene.

Interaction of the proteasome S5a/Rpn10 multiubiquitin-binding protein and the 8 kDa calcium-binding protein of Schistosoma mansoni.

A distinct 8 kDa calcium-binding protein (CaBP) is preferentially expressed at the cercarial stage during the life-cycle of the schistosome. Available data indicate that this CaBP may be associated with tissue/organ remodelling (involving protein degradation and synthesis of new proteins) during transformation of the cercariae from free-living form in water to parasitic life in the vertebrate host. Many CaBP molecules (e.g. calmodulin) show Ca(++)-dependent interaction with target proteins and thus modulate their activity. Accordingly, the parasite 8 kDa CaBP was used as a probe to clone and identify putative target protein(s) directly by binding interaction. Screening of schistosome lambdagt11 expression library with radio-iodinated CaBP yielded several overlapping clones showing Ca(++)-dependent binding of the CaBP. Sequence analyses revealed that these clones encode the S5a/Rpn10 multiubiquitin-binding protein which is a component of the regulatory 19S subunit of the 26S proteasome. The schistosome molecule, designated SmS5a, is 420 amino acids long. The nearly full length molecule (Gln3-Ser420) as well as the amino terminal (N-S5a, Gln3-Gly200) and carboxyl-terminal (C-S5a, Asp225-Ser420) portions were synthesized in bacteria, purified, and antibodies to the parasite SmS5a were prepared. Interaction between SmS5a and the 8 kDa CaBP in a Ca(++)-dependent manner was found under various experimental conditions: CaBP-Sepharose bound soluble SmS5a, immobilized SmS5a bound soluble CaBP, and complex formation was found when both molecules were in solution. Furthermore, it was shown that the C-terminal portion of SmS5a, but not the N-terminal portion of the molecule, reacted with the CaBP. SmS5a synthesized in a cell-free system and Western blots revealed 2 species, conceivably corresponding to the naked molecule (approximately 50 kDa) and the molecule subjected to post-translational modification (approximately 70 kDa). The present studies suggest that proteasome activity may be modulated by calcium, and this modulation is mediated via CaBP molecule(s).

Stage-specific alternative splicing of the heat-shock transcription factor during the life-cycle of Schistosoma mansoni.

Stage-specific alternative splicing of the heat-shock transcription factor of Schistosoma mansoni (SmHSF) generates isoforms with structural diversity that may modulate the activity of SmHSF at different life-stages, and thus may regulate the expression of different genes at different developmental stages. RT-PCR, cloning and DNA-sequence analyses showed stage-specific alternative splicing inside the DNA-binding domain (DBD) involving introns I1 and I2, and beyond the DBD involving introns I4a and I7. Retention of introns I2 and I4 would inactivate SmHSF since they contain termination codons. Retention of intron I1 would add 11 amino acids inside the DBD and may change the DNA-binding specificity of SmHSF; intron I7 would add 13 amino acids to the effector region of HSF. Retention of introns was more pronounced in cercariae (larval stage living in water) than in adult worms (parasitic form in mammals). The isoforms were expressed in bacteria, but functional evaluation was not feasible, because only the isoform lacking introns was soluble while isoforms with introns were insoluble. However, stage-specific alternative splicing that changed HSF function in vivo was evidenced in intact cercariae. The cercarial SmHSF mRNA was enriched with introns I2 and I4a that contain termination codons. Therefore, translation of the SmHSF mRNA was impaired, and the SmHSF protein was undetectable. Consequently, the HSP70 gene could not be transcribed, and the HSP70 mRNA was missing. Alternative splicing was observed for short DNA segments (33-45 bp) bound by splice signals, located in the coding region. These are not bona fida exons since they are not flanked by introns. Yet, they are not regular introns since they are often found in mature mRNA. Alternative splicing of these DNA segments caused structural diversity that could modulate the function of the gene product.

Somatic diversification of chicken immunoglobulin light chains by point mutations.

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).

Low levels of IFN-gamma down-regulate the integrin-dependent adhesion of B cells by activating a pathway that interferes with cytoskeleton rearrangement.

In order to fully mature and participate in the humoral immune response, immature B cells must first migrate into specific areas in the spleen where they differentiate into mature cells. However, before their maturation in the spleen, immature B cells must be excluded from non-splenic secondary lymphoid organs where any antigen encounter would lead to the death of the cells because of the negative selection process. We have recently shown that immature B cells can actively exclude themselves from antigen-enriched sites by down-regulating their integrin-mediated adhesion in a process mediated by interferon-gamma (IFN-gamma). In this study, we followed the pathway by which IFN-gamma regulates the homing of B cells. We show here that the inhibitory signal of IFN-gamma is transmitted through the IFN-gamma receptor whose engagement leads to the activation of PI3K. This PI3K activation subsequently leads to the inhibition of PKCalpha phosphorylation and cytoskeleton rearrangement required for promoting integrin-mediated adhesion and migration of B cells.

Schistosoma mansoni: stage-specific expression of muscle-specific genes.

It was previously shown that an antigen preparation termed 9B obtained from Schistosoma mansoni cercarial extracts partially (34%) protects mice from challenge infection with cercariae (R. Tarrab-Hazdai et al., J. Immunol. 135, 2772, 1985). To characterize some of the proteins which comprise this preparation, rabbit antibodies to the 9B antigen preparation were used to screen cDNA libraries of cercariae and adult worms. We isolated and sequenced cDNA clones encoding three proteins: calcium-binding protein, paramyosin, and myosin. The calcium-binding protein was previously shown to be expressed in cercariae but not in sporocysts or adult worms (D. Ram et al., Mol. Biochem. Parasitol. 34, 167, 1989). Northern blots showed the presence of paramyosin and myosin mRNAs in sporocysts and adult worms but not in cercariae. Antibodies to paramyosin detected the protein in sporocysts and adult worms as well as in cercariae. These findings explain, in part, the protective activity of the 9B antigen preparation against challenge infection.