Bacterial challenge stimulates innate immune responses in extra-embryonic tissues of tobacco hornworm eggs.

Innate immunity protects juvenile and adult vertebrates and invertebrates against potential pathogens; however, it is unknown when developing embryos become immune competent and just how they are guarded from infection. To address these questions, we studied the effect of immune challenge on early stage eggs of the tobacco hornworm, Manduca sexta. We detected many immune-related proteins and mRNAs in naive eggs. Upon immune challenge, antimicrobial protein genes were up-regulated, and antibacterial activity increased. Antimicrobial protein mRNAs and lysozyme were present in the extra-embryonic tissues of immune-challenged eggs; in addition, melanization in response to bacteria occurred in the yolk but not embryonic tissues. We conclude that the extra-embryonic tissues of early stage M. sexta eggs are immune competent and likely protect the developing embryo from infection. We suggest that innate immune responses of extra-embryonic tissues may be a common mechanism for protecting early embryos.

Serine proteases as mediators of mosquito immune responses.

Serine proteases regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization of pathogen surfaces. These processes require the presence of serine proteases in the hemolymph where they can rapidly activate immune pathways in response to pathogen detection. Hemolymph coagulation in the horseshoe crab is controlled by several serine proteases, including two that are pathogen recognition molecules and two in the clip domain family of serine proteases. The antimicrobial peptide synthesis and melanization pathways include clip domain proteases as well as other, uncharacterized serine proteases. We have identified five serine proteases from the hemolymph of the mosquito, Anopheles gambiae. One, Sp22D, is a large protease with potential pathogen binding domains. Sp22D is expressed in three tissues that have immune functions (midgut epithelium, fat body, and hemocytes), and its transcript abundance increases after immune challenge. Sp14A, Sp14D1, and Sp14D2 are clip domain serine proteases that are similar to enzymes with presumed roles in melanization or antimicrobial peptide synthesis. They undergo changes in transcript abundance in response to infection with bacteria or malaria parasites, and they reside in a chromosomal region that has been associated with melanization of parasites. Sp18D, also a clip domain protease, is similar to a Manduca protease with a likely role in immunity, but immune challenge does not affect its mRNA abundance.

Sp22D: a multidomain serine protease with a putative role in insect immunity.

Serine proteases play critical roles in a variety of insect immune responses; however, few of the genes that code for these enzymes have been cloned. Here, we describe the molecular characterization of a serine protease gene from the mosquito Anopheles gambiae. Sp22D codes for a 1322 amino acid polypeptide with a complex domain organization. In addition to the carboxy terminal serine protease catalytic domain, Sp22D contains two putative chitin binding domains, a mucin-like domain, two low density lipoprotein receptor class A domains, and two scavenger receptor cysteine rich domains. A typical signal peptide sequence and a lack of potential transmembrane helices suggest that Sp22D is secreted. Sp22D is expressed constitutively in three immune-related cell types: adult hemocytes, fat body cells, and midgut epithelial cells. Wounding induces no changes in transcript abundance, but within 1h after injection of bacteria, Sp22D mRNA increases 1.5-fold. Based on domain organization, tissue distribution, and transcriptional up-regulation in response to immune challenge, we suggest that Sp22D has an immune function. In addition, we predict that Sp22D is secreted into the hemolymph where it may interact with pathogen surfaces and initiate an immune response.

Intravenous tissue plasminogen activator for acute ischemic stroke in patients aged 80 years and older : the tPA stroke survey experience.

BACKGROUND AND PURPOSE: Intravenous tissue plasminogen activator (tPA) administered within 3 hours of symptom onset is the first available effective therapy for acute ischemic stroke (AIS). Few data exist, however, on its use in very elderly patients. We examined the characteristics, complications, and short-term outcome of AIS patients aged >/=80 years treated with tPA. METHODS: Patients aged >/=80 years (n=30) were compared with counterparts aged <80 years (n=159) included in the tPA Stroke Survey, a US retrospective survey of 189 consecutive AIS patients treated with intravenous tPA at 13 hospitals. RESULTS: Risk of intracerebral hemorrhage (fatal, symptomatic, and total) was 3%, 3%, and 7% in the elderly age group and 2%, 6%, and 9%, respectively, in their younger counterparts (P=NS for all comparisons). Likelihood of favorable outcome, defined as modified Rankin score 0 to 1, National Institutes of Health Stroke Scale score /=80 years was identified.

Molecular characterization of five serine protease genes cloned from Anopheles gambiae hemolymph.

We identified five new serine protease cDNAs from the hemolymph of the malaria vector, Anopheles gambiae. All five show sequence similarity to genes thought to be involved in vertebrate or invertebrate defense responses. Sp14A, Sp14D2 and Sp22D demonstrate changes in transcript abundance in response to bacteria injections. Sp14A and Sp14D2, as well as the previously characterized Sp14D1, are induced by infection with the malaria parasite, Plasmodium berghei. These three proteases, along with Sp18D, are related to a group of secreted proteases that have amino-terminal clip domains and trypsin-like substrate specificity. BLAST results and phylogenetic analyses group Sp14A, Sp14D1 and Sp14D2 with the Drosophila protease EASTER, and three prophenoloxidase activating enzymes from other insects. EASTER's substrate is SPAETZLE, a ligand involved in embryogenesis but also in activating anti-microbial peptide synthesis. Their similarity to EASTER and immune inducibility suggest that one of these proteases may activate a SPAETZLE-like ligand during anti-parasite responses in mosquitoes. Alternatively, as potential prophenoloxidase activators, Sp14A, Sp14D1 or Sp14D2 may play a role in melanotic encapsulation of Plasmodium.

An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta. The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes. The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab. In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5' end of the gene confirms the single copy status of this gene. Northern analysis reveals changes in transcript abundance during development and following blood feeding. Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.

Ataxic hemiparesis: critical appraisal of a lacunar syndrome.

BACKGROUND AND PURPOSE: Ataxic hemiparesis is a well-recognized lacunar syndrome involving homolateral ataxia with accompanying corticospinal tract impairment. Despite 30 years of clinical experience there continues to be some doubt as to the defining clinical characteristics, precise neuroanatomic localization of the syndrome, and etiologic mechanisms. METHODS: We now present 45 new cases that have been analyzed for clinico-radiologic correlation and etiology. Also, all published cases from the English literature known to the authors are reviewed. RESULTS: We found that the clinical syndrome of ataxic hemiparesis accurately predicts a small deep infarction, generally in the pons or internal capsule. Sensory loss is highly associated with a capsular localization. We found that 47% of the cases were attributed to small-vessel disease, 11% to cardioembolism, and only 7% to artery-to-artery embolism (all in the basilar artery); 1 case was attributed to thrombocytosis, 1 to multiple sclerosis, and the rest either had negative or incomplete evaluation. Approximately two thirds of the infarctions occurred in patients with neuroimaging evidence of other ischemic brain lesions. CONCLUSIONS: Ataxic hemiparesis is a distinct clinical syndrome that accurately predicts a small deep infarction, most commonly in the pons or internal capsule. Only sensory loss accurately predicts a capsular localization. Etiology in nearly half of the cases can be attributed to small-vessel disease. Furthermore, ataxic hemiparesis appears to be a good marker for generalized asymptomatic cerebrovascular disease.

The role of surface characteristics in eliciting humoral encapsulation of foreign bodies in Plasmodium-refractory and -susceptible strains of Anopheles gambiae.

A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and kills many species of malaria parasites, whereas susceptible strains allow the parasites to develop normally. To study the role of surface characteristics in eliciting this immune response, 27 types of chromatography beads that differed in matrix type, charge, functional group, and functional group density were assayed for degree of melanotic encapsulation in refractory and susceptible mosquitoes. Overall, two glucan-based matrices, Sephadex (dextran) and cellulose, stimulated the strongest responses, regardless of functional group. Substituting matrix hydroxyl groups with functional groups on Sephadex and cellulose beads decreased the level of encapsulation. These results demonstrate that glucans induce melanotic encapsulation in An. gambiae. Beads with agarose, polystyrene, and acrylic matrices, and most methacrylate-based beads elicited little or no melanization; however, epoxide-methacrylate beads were encapsulated, demonstrating that glucans are not essential for eliciting a response. Comparisons between the two strains demonstrated that refractory mosquitoes melanized many bead types to a greater degree than did susceptible mosquitoes. On this basis, we propose that an important difference between the two strains is that one of the enzymes involved in the melanization pathway functions at a higher level in the refractory strain. Finally, of all beads tested, only 85% substituted CM-Sephadex beads were virtually unmelanized in susceptible mosquitoes but highly melanized in the refractory strain; thus, a specific surface microenvironment is necessary to demonstrate this effect.

Cloning and characterization of a serine protease from the human malaria vector, Anopheles gambiae.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp24D) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 271 amino acid protein that contains the conserved serine, histidine and aspartic acid residues found in serine proteases, and has the highest identity to a serine protease of unknown function from Drosophila melanogaster. In situ hybridization to the polytene chromosomes detects a single band at 24D. Northern analysis reveals only low levels of transcripts in larvae and pupae, but more abundant transcription products occur in adults. Interestingly, this analysis also shows that adult males express much higher levels of AgSp24D mRNA than females. In addition, Plasmodium-refractory mosquitoes express higher levels of AgSp24D mRNA than susceptible mosquitoes although the biological significance of this remains to be examined. The thorax is the primary site for expression in the adults. The lack of a dramatic increase in AgSp24D mRNA levels following blood feeding suggests that this protease is not involved in digestive processes. Transcriptional induction does not follow cold shock, septic wounding, bacterial injection, laminarin injection or CM-Sephadex bead injection.

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

A genetic study of a melanization response to Sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae.

A previously selected Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium. Genetic studies of this strain have shown that this refractory phenotype is controlled by a limited number of genes, and the existence of two such genes, Pif-B and Pif-C, has been demonstrated. Further work to determine the molecular basis for this mode of refractoriness led to the discovery that the host-parasite interaction is mimicked by the mosquito's response to carboxymethyl (CM)-Sephadex beads injected into the thorax. These small beads are melanized within 24 hr in refractory mosquitoes but are rarely melanized in susceptible ones. Because of the considerable potential in using bead melanization as a model for Plasmodium refractoriness, we performed a genetic analysis of the differential response to beads. Reciprocal crosses of susceptible (4arr) and refractory (L35) mosquitoes and an analysis of F1 phenotypes were done. The F1 progeny had a phenotype similar to that of the parental refractory mosquitoes; therefore, dominant refractory allele(s) must be present in the refractory strain. Males from the reciprocal crosses had identical phenotypes, indicating that X-linked loci did not have a visible effect on the melanizing phenotype. To further study the mode of inheritance of the melanizing trait, a backcross of F1 females to 4arr males was done. The phenotypic distribution of the backcross progeny was bimodal, and the melanization phenotypes were similar to those of the susceptible and refractory parents. These data suggest that a small number of loci are responsible for the differential response to CM-Sephadex beads, and that one chromosomal region contributes strongly to the melanizing trait. Because the 4arr strain carries mutations in the pink eye (X) and red eye (III) genes, possible linkage of the melanizing phenotype to these two genes was tested. No linkage with either marker was detected. The pattern of inheritance of the melanizing phenotype is similar to that of the refractory phenotype of Pif-B; therefore, the genetic basis of the two responses may be the same.

A shared genetic mechanism for melanotic encapsulation of CM-Sephadex beads and a malaria parasite, Plasmodium cynomolgi B, in the mosquito, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae that melanizes ookinetes and intrathoracically inoculated CM-Sephadex beads was mated to a Plasmodium-susceptible strain that does not melanize the parasite or the beads. The F1 progeny were then backcrossed to the susceptible strain. Backcross progeny were given a blood meal containing infective Plasmodium cynomolgi B, and the parasites were allowed to develop for 6-7 days, at which time the infected mosquitoes were injected with CM-Sephadex beads. The next day the mosquitoes were dissected and the beads were scored for degree of melanization while the parasites were scored for degree of encapsulation. A Spearman rank order correlation test of the degree of correlation between the bead melanization phenotype and the parasite encapsulation phenotype gave a correlation coefficient of 0.74 (P < 0.01). This strong correlation between the two melanization responses suggests that the mechanisms for differential bead and parasite melanization of these two mosquito strains share at least one major gene.

Genetic analysis of embryonic cis-acting regulatory elements of the Drosophila homeotic gene sex combs reduced.

The homeotic gene Sex combs reduced (Scr) of Drosophila melanogaster is expressed in the labial and prothoracic segments of the ectoderm, in parasegments two and three of the CNS, and in the visceral mesoderm of the anterior and posterior midgut. The mutationally defined function of Scr is to specify the identity of the labial and prothoracic segments and to control the development of the gastric caeca. The Scr locus occupies a chromosomal region of approximately 80 kb within the Antennapedia complex (ANT-C). To understand how Scr's spatiotemporal expression pattern is generated in the embryo, we have mapped its transcriptional regulatory elements using three approaches. First, we examined the expression pattern of Scr in embryos containing chromosomal rearrangements that remove potential Scr regulatory elements. Second, we made and analyzed a set of Scr minigene transformants. Third, we analyzed a set of Scr-lacZ enhancer tester constructs. Using more sensitive anti-SCR antisera, we discovered that Scr is expressed in tissues that were not previously thought to accumulate SCR: a stripe of ectodermal cells in the parasegment 2 region of stage 5 embryos, the embryonic salivary glands, and the dorsal ridge. Four DNA fragments that had previously been shown in an analysis of Scr-lacZ reporter constructs to contain putative Scr enhancer elements were found to have functional enhancers; similarly, another Scr fragment was found to contain a functional repressor. Our results suggest that regulation of Scr in the labial segment and the CNS requires the apparently synergistic action of multiple, widely spaced enhancer elements. Regulation in the prothorax also appears to be controlled by multiple enhancers:one complete pattern element and one subpattern element. In contrast, Scr regulation in the visceral mesoderm is controlled by an enhancer(s) located in only one DNA fragment.

A genetic analysis of deltex and its interaction with the Notch locus in Drosophila melanogaster.

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.