Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase.

Lipoprotein lipase (LPL) is a key enzyme in the hydrolysis of triglyceride-rich lipoproteins. Conflicting results have been reported concerning its role in atherogenesis. To determine the effects of the overexpressed LPL on diet-induced atherosclerosis, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpressed human LPL transgene (LPL/LDLRKO) and compared their plasma lipoproteins and atherosclerosis with those in nonexpressing LDLR-knockout mice (LDLRKO). On a normal chow diet, LPL/LDLRKO mice showed marked suppression of mean plasma triglyceride levels (32 versus 236 mg/dl) and modest decrease in mean cholesterol levels (300 versus 386 mg/dl) as compared with LDLRKO mice. Larger lipoprotein particles of intermediate density lipoprotein (IDL)/LDL were selectively reduced in LPL/LDLRKO mice. On an atherogenic diet, both mice exhibited severe hypercholesterolemia. But, mean plasma cholesterol levels in LPL/ LDLRKO mice were still suppressed as compared with that in LDLRKO mice (1357 versus 2187 mg/dl). Marked reduction in a larger subfraction of IDL/LDL, which conceivably corresponds to remnant lipoproteins, was observed in the LPL/LDLRKO mice. LDLRKO mice developed severe fatty streak lesions in the aortic sinus after feeding with the atherogenic diet for 8 weeks. In contrast, mean lesion area in the LPL/LDLRKO mice was 18-fold smaller than that in LDLRKO mice. We suggest that the altered lipoprotein profile, in particular the reduced level of remnant lipoproteins, is mainly responsible for the protection by LPL against atherosclerosis.

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Attenuated virulence of chitin-deficient mutants of Candida albicans.

We have analyzed the role of chitin, a cell-wall polysaccharide, in the virulence of Candida albicans. Mutants with a 5-fold reduction in chitin were obtained in two ways: (i) by selecting mutants resistant to Calcofluor, a fluorescent dye that binds to chitin and inhibits growth, and (ii) by disrupting CHS3, the C. albicans homolog of CSD2/CAL1/DIT101/KT12, a Saccharomyces cerevisiae gene required for synthesis of approximately 90% of the cell-wall chitin. Chitin-deficient mutants have no obvious alterations in growth rate, sugar assimilation, chlamydospore formation, or germ-tube formation in various media. When growing vegetatively in liquid media, the mutants tend to clump and display minor changes in morphology. Staining of cells with the fluorescent dye Calcofluor indicates that CHS3 is required for synthesis of the chitin rings found on the surface of yeast cells but not formation of septa in either yeast cells or germ tubes. Despite their relatively normal growth, the mutants are significantly less virulent than the parental strain in both immunocompetent and immunosuppressed mice; at 13 days after infection, survival was 95% in immunocompetent mice that received chs3/chs3 cells and 10% in immunocompetent mice that received an equal dose of chs3/CHS3 cells. Chitin-deficient strains can colonize the organs of infected mice, suggesting that the reduced virulence of the mutants is not due to accelerated clearing.

A mutation in the promoter of the lipoprotein lipase (LPL) gene in a patient with familial combined hyperlipidemia and low LPL activity.

We have identified a naturally occurring mutation in the promoter of the lipoprotein lipase (LPL) gene. One of 20 patients with familial combined hyperlipidemia (FCHL) and reduced levels of postheparin plasma LPL activity was found to be a heterozygote carrier of this mutation. The mutation, a T-->C substitution at nt -39, occurred in the binding site of the transcription factor Oct-1. As a result, the transcriptional activity of the mutant promoter was < 15% of wild type, as determined by transfection studies in the human macrophage-like cell line THP-1. This decrease in promoter activity was observed in undifferentiated as well as in phorbol ester-differentiated THP-1 cells. Furthermore, the inductive effect of elevating the levels of intracellular cAMP was equally reduced. This mutation was not present among 20 FCHL patients with normal plasma LPL levels nor has it been reported among individuals with familial LPL deficiency. Thus, heterozygosity for LPL promoter mutations may be one of several factors that contribute to the etiology of FCHL.

Molecular cloning and characterization of chitinase genes from Candida albicans.

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.

Candida albicans stimulates in vivo differentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2-dependent signalling

Toll-like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2-dependent signalling. Purified lineage-negative cells (Lin−) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin− cells from TLR2−/− mice did not give rise to macrophages, whereas Lin− cells from TLR4−/− mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin− cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR-mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR-mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.

Role of IFN-gamma in immune responses to Candida albicans infections

Candida albicans is the most frequent etiologic agent that causes opportunistic fungal infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-γ) in host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions leading to the production of IFN-γ by host cells. IFN-γ is produced by CD4 Th1, CD8, γδ T, and natural killer (NK) cells, essentially in response to both IL-12 and/or IL-18; more recently, a subset of C. albicans-specific Th17 cells have been described to produce both IL-17 and IFN-γ. IFN-γ plays an important role in the regulation of the immune system as well as in the control of the infectious process, as it is required for optimal activation of phagocytes, collaborates in the generation of protective antibody response, and favors the development of a Th1 protective response.

Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

Will Familial Hypercholesterolaemia Cohorts Hide Many More Lisosomal Acid Lipase Deficiency Patients?

Aims: Lisosomal Acid Lipase Deficiency (LALD), historical known as Cholesterol Ester Storage Disease (CESD), is an autosomal lisosomal storage recessive disorder and an unrecognized cause of dyslipidaemia. Mutations in LIPA gene are the underlying cause of LALD, being a mutation in the splice site of exon 8 the most common cause of the disease. Patients with LALD present dyslipidaemia and altered liver function. The aim of this work was to analyze LIPA gene in patients with unexplained dyslipidaemia.

Immune recovery in HIV-infected patients after candida esophagitis is impaired despite long-term antiretroviral therapy.

OBJECTIVE Candida esophagitis belongs to the most common AIDS-defining diseases, however, a comprehensive immune pathogenic concept is lacking. DESIGN We investigated the immune status of 37 HIV-1-infected patients from the Swiss HIV cohort study at diagnosis of Candida esophagitis, 1 year before, 1 year later and after 2 years of suppressed HIV RNA. We compared these patients to 3 groups: 37 HIV-1-infected patients without Candida esophagitis but similar CD4 counts as the patients at diagnosis (advanced HIV group), 15 HIV-1-infected patients with CD4 counts >500 cells/μl, CD4 nadir >350 cells/μl and suppressed HIV RNA under combination antiretroviral therapy (cART) (early cART group), and 20 healthy individuals. METHODS We investigated phenotype, cytokine production and proliferative capacity of different immune cells by flow cytometry and ELISpot. RESULTS We found that patients with Candida esophagitis had nearly abolished CD4 proliferation in response to C. albicans, significantly increased percentages of dysfunctional CD4 cells, significantly decreased cytotoxic NK-cell counts and peripheral innate lymphoid cells and significantly reduced IFN-γ and IL-17 production compared to the early cART group and healthy individuals. Most of these defects remained for more than 2 years despite viral suppression. The advanced HIV group without opportunistic infection showed partly improved immune recovery. CONCLUSIONS Our data indicate that Candida esophagitis in HIV-1-infected patients is caused by an accumulation of multiple, partly Candida-specific immunological defects. Long-term immune recovery is impaired, illustrating that specific immunological gaps persist despite cART. These data also support the rationale for early cART initiation to prevent irreversible immune defects.

Solennizzandosi il giorno XII agosto MDCCCXLIV nel convento delle Clarisse in Lovere la professione di suor Maria Candida Rosa Molinari : canzone : dedicata al merito esimio del signore Giovita Cantoni.

Acquisition made accessible thanks to a 2015-2017 grant from the Council on Libraries and Information Resources.

Pronunciando i voti solenni suor Maria Candida Molinari nel monastero delle Clarisse in Lovere : sonetto : dedicato al merito esimio del molto rev. signore don Pietro Superti, degnissimo rettore del Santuario delle Grazie in Brescia.

Acquisition made accessible thanks to a 2015-2017 grant from the Council on Libraries and Information Resources.