Stimulus-dependent impairment of the neutrophil oxidative burst response in lactoferrin-deficient mice.

Lactoferrin (LF) is an iron-binding protein found in milk, mucosal secretions, and the secondary granules of neutrophils in which it is considered to be an important factor in the innate immune response against microbial infections. Moreover, LF deficiency in the secondary granules of neutrophils has long been speculated to contribute directly to the hypersusceptibility of specific granule deficiency (SGD) patients to severe, life-threatening bacterial infections. However, the exact physiological significance of LF in neutrophil-mediated host defense mechanisms remains controversial and has not yet been clearly established in vivo using relevant animal models. In this study, we used lactoferrin knockout (LFKO) mice to directly address the selective role of LF in the host defense response of neutrophils and to determine its contribution, if any, to the phenotype of SGD. Neutrophil maturation, migration, phagocytosis, granule release, and antimicrobial response to bacterial challenge were unaffected in LFKO mice. Interestingly, a stimulus-dependent defect in the oxidative burst response of LFKO neutrophils was observed in that normal activation was seen in response to opsonized bacteria whereas an impaired response was evident after phorbol myristate-13-acetate stimulation. Taken together, these results indicate that although LF deficiency alone is not a primary cause of the defects associated with SGD, this protein does play an immunomodulatory role in the oxidative burst response of neutrophils.

VSGdb: a database for trypanosome variant surface glycoproteins, a large and diverse family of coiled coil proteins.

BACKGROUND: Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long alpha-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for alpha helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences. DESCRIPTION: VSGdb contains fully annotated VSG sequences from the genome sequencing project, with which it shares all identifiers and annotation, and other available sequences. The database can be queried in various ways. Sequence retrieval, in FASTA format, can deliver protein or nucleotide sequence filtered by chromosomes or contigs, gene type (functional, pseudogene, etc.), domain and domain sequence family. Retrieved sequences can be stored as a temporary database for BLAST querying, reports from which include hyperlinks to the genome project database (GeneDB) CDS Info and to individual VSGdb pages for each VSG, containing annotation and sequence data. Queries (text search) with specific annotation terms yield a list of relevant VSGs, displayed as identifiers leading again to individual VSG web pages. CONCLUSION: VSGdb http://www.vsgdb.org/ is a freely available, web-based platform enabling easy retrieval, via various filters, of sets of VSGs that will enable detailed analysis of a number of general and trypanosome-specific questions, regarding protein structure potential, epitope variability, sequence evolution and recombination events.

Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi.

BACKGROUND: The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids. RESULTS: Bioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs. CONCLUSION: Trypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules.

Delayed vasodilatory response to methylnicotinate in patients with unipolar depressive disorder.

BACKGROUND: Recent evidence has suggested an important role for lipids in the etiology and treatment of depression. Methylnicotinate-induced vasodilation can be used to investigate lipid-dependent signalling mechanisms involving the phospholipase A2 (PLA2)/cyclooxygenase pathway, an important signalling system involved in the action of several neurotransmitters including serotonin. To investigate whether abnormalities in this signalling system may occur in depressive illness, we undertook a study of methylnicotinate response in unipolar depression (UD). METHODS: Methylnicotinate was applied to the forearm of 20 patients with depression and 38 age and sex-matched healthy volunteers (HV). The resulting erythema was assessed over a 15-min period. RESULTS: Methylnicotinate-induced erythema was reduced in subjects with depression compared to HV at 5 min after application, it returned to normal after 15 min. Thus, although the maximal response to methylnicotinate appears normal, patients with UD exhibit an apparently delayed response. LIMITATIONS: The major limitation is that all unipolar patients were medicated at the time of testing. CONCLUSIONS: Our results support the hypothesis that UD may be associated with abnormalities in lipid-associated signalling systems, and may provide insight into how lipid intake may modulate depressive symptoms.

Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote.

BACKGROUND: Malaria, caused by the parasitic protist Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism. RESULTS: Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in P. falciparum and just one member in some other Apicomplexan species. CONCLUSION: The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells.

Lactoferrin: role in iron homeostasis and host defense against microbial infection.

The transferrin family of non-heme iron binding glycoproteins are believed to play a central role in iron metabolism and have been implicated in iron transport, cellular iron delivery and control of the level of free iron in external secretions. Lactoferrin (LF) is a member of this family that is widely localized in external fluids including milk and mucosal secretions, in addition to being a prominent component of the secondary granules of neutrophils. Although structurally related to transferrin, LF appears to have a broader functional role mediated by both iron dependent and iron independent mechanisms. In this review, we will focus on our current understanding on the role of LF in regulating iron homeostasis and its role in host protection against microbial infection at the mucosal surface. In addition, recent insights obtained from analyzing the phenotypic consequences of LF ablation in lactoferrin knockout mice (LFKO), which challenge the long held dogma that LF is required for intestinal iron absorption in the neonate, are summarized.

The investigation of cytosolic phospholipase A2 using ELISA.

Two studies of the behaviour of cytosolic phospholipase A(2) (cPLA(2)) in the red blood cell (RBC), as measured by ELISA, are described. In the first study we show a significant increase in cPLA(2) in patients with schizophrenia compared to controls and suggest that this measure, if corroborated, could be used as a diagnostic marker. In a second study we found that washing the RBC introduced an unknown confounding variable which led us to reject this study. A subsequent investigation of washing red cells showed that the washing effect may be due to a plasma factor likely to be more than 5kDa MW which can be removed from red cells by washing with buffers. When the cells are washed, the concentration of cPLA(2) in the red cell, as measured by ELISA, significantly increases. We advise against washing the red cell in any study that involves measuring cPLA(2) by ELISA.

On the relationship between methylnicotinate-induced skin flush and fatty acids levels in acute psychosis.

Vasodilation induced by methylnicotinate, a fatty acid- and cyclooxygenase-dependent process, is reduced or absent in patients with schizophrenia. This phenomenon has been suggested to be useful as a diagnostic test for the illness. To determine whether reduced flushing is specific to schizophrenia and is caused by a deficiency in membrane fatty acids, the extent of topically applied methylnicotinate-induced vasodilation was measured in 23 subjects with schizophrenia, 20 subjects with bipolar disorder and 34 healthy volunteers along with red cell fatty acid concentrations and measures of clinical severity. Although there was a significant decrease in an estimate of vasodilation (erythema) compared with healthy volunteers in both schizophrenia and bipolar groups, the schizophrenia group responded significantly less than subjects with bipolar disorder. The reduction in the bipolar group was partly due to a delayed vasodilatory reaction, an effect not observed in subjects with schizophrenia. In subjects with schizophrenia, there were no significant correlations between methylnicotinate response and fatty acid concentrations. The authors conclude that the methylnicotinate procedure can differentiate schizophrenia from other serious mental illness. The methylnicotinate insensitivity in schizophrenia, however, is likely to be due to a deficiency in the fatty acid precursors required for the vasodilatory reaction.

Iron status in mice carrying a targeted disruption of lactoferrin.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO(-/-)) mice by homologous gene targeting. LFKO(-/-) mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO(-/-) intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO(-/-) mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

Lactoferrin and host defense.

Lactoferrin is a multifunctional member of the transferrin family of nonheme iron-binding glycoproteins. Lactoferrin is found at the mucosal surface where it functions as a prominent component of the first line of host defense against infection and inflammation. The protein is also an abundant component of the specific granules of neutrophils and can be released into the serum upon neutrophil degranulation. While the iron-binding properties were originally believed to be solely responsible for the host defense properties ascribed to lactoferrin, it is now known that other mechanisms contribute to the broad spectrum anti-infective and anti-inflammatory roles of this protein. In this article, current information on the functions and mechanism of action of lactoferrin are reviewed, with particular emphasis on the activities that contribute to this protein's role in host defense. In addition, studies demonstrating that lactoferrin inhibits allergen-induced skin inflammation in both mice and humans, most likely secondary to TNF-alpha (tumor necrosis factor alpha) production, are summarized. Collectively, these results suggest that lactoferrin functions as a key component of mammalian host defense at the mucosal surface.