Heritable induced resistance in Arabidopsis thaliana: Tips and tools to improve effect size and reproducibility.

Over a decade ago, three independent studies reported that pathogen- and herbivore-exposed Arabidopsis thaliana produces primed progeny with increased resistance. Since then, heritable induced resistance (h-IR) has been reported across numerous plant-biotic interactions, revealing a regulatory function of DNA (de)methylation dynamics. However, the identity of the epi-alleles controlling h-IR and the mechanisms by which they prime defense genes remain unknown, while the evolutionary significance of the response requires confirmation. Progress has been hampered by the relatively high variability, low effect size, and sometimes poor reproducibility of h-IR, as is exemplified by a recent study that failed to reproduce h-IR in A. thaliana by Pseudomonas syringae pv. tomato (Pst). This study aimed to improve h-IR effect size and reproducibility in the A. thaliana-Pst interaction. We show that recurrent Pst inoculations of seedlings result in stronger h-IR than repeated inoculations of older plants and that disease-related growth repression in the parents is a reliable marker for h-IR effect size in F1 progeny. Furthermore, RT-qPCR-based expression profiling of genes controlling DNA methylation maintenance revealed that the elicitation of strong h-IR upon seedling inoculations is marked by reduced expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) gene, which is maintained in the apical meristem and transmitted to F1 progeny. Two additional genes, MET1 and CHROMOMETHYLASE3 (CMT3), displayed similar transcriptional repression in progeny from seedling-inoculated plants. Thus, reduced expression of DDM1, MET1, and CMT3 can serve as a marker of robust h-IR in F1 progeny. Our report offers valuable information and markers to improve the effect size and reproducibility of h-IR in the A. thaliana-Pst model interaction.

Antigen-driven C-C chemokine-mediated HIV-1 suppression by CD4(+) T cells from exposed uninfected individuals expressing the wild-type CCR-5 allele.

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1-specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32-base pair deletion in the C-C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Delta32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C-C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell-tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development.

[Autosomal dominant polycystic kidney disease: from genes to cilium]. / Rene policistico autosomico dominante: dai geni al cilio.

Autosomal dominant polycystic kidney disease (ADPKD) is a quite frequent monogenic hereditary disease. The incidence has been reported to range between 1:400 and 1:1000 life births. The disease is caused by a mutation of the PKD1 gene in 85% of the cases and by a mutation of the PKD2 gene in the remaining 15%. The main characteristic of this condition is the development of renal cysts. Observations regarding various cystic kidney diseases sustained by mutations of different genes are steadily converging to a common point. This unifying element is the primary cilium. The cilium, which has long been considered a mere biological oddity, has lately become the focus of intense scientific attention because it may turn out to be the key to the understanding of cystic degeneration. The cilia can be regarded as sensors projecting out of the cell. In particular in the kidney they are located in an ideal place to capture information from the tubular lumen. One of the roles the cilia may play is the reception of chemical signals. An alternative hypothesis attributes to the cilia the role of mechanosensors capable of detecting variations of the urine flux in the tubular lumen. The cilium projects itself into the lumen where it can readily capture variations in the external environment and transmit them to the cell by as yet undefined pathways. This is the still largely unexplored frontier that will provide the elements needed to understand and treat renal cystic diseases.

Infection of circulating CD34+ cells by HHV-8 in patients with Kaposi's sarcoma.

Human herpesvirus type 8 (HHV-8) has been identified as the most likely candidate to be involved in the development of Kaposi's Sarcoma (KS). HHV-8 has been associated with all forms of KS, primary effusion lymphoma, and multicentric Castleman's disease and detected in various non-neoplastic cells. Its presence in cells of the different hemopoietic lineages has not yet been investigated in a comprehensive and systematic manner. In this study we searched for the presence of HHV-8 in different subpopulations of peripheral blood mononuclear cells (PBMC) from patients with classic and AIDS-associated KS, as well as from HIV-1 sero-positive and sero-negative persons without KS. Thirty-four samples of PBMC were isolated from 30 patients. Subpopulations were isolated with immunomagnetic beads. Polymerase chain reaction for HHV-8 DNA was performed on PBMC and subpopulations with a primer pair selected from ORF26 of the viral genome. Polymerase chain reaction products were subsequently Southern blotted and hybridized. In patients with KS, HHV-8 DNA was detected in nine of 11 (81%) CD19+ cells, four of 11 (36%) CD2+ cells, three of 11 (27%) CD14+ cells, and nine of 11 (81%) of the remaining depleted cell populations (DP) that contain CD34 positive cells. In a subsequent set of experiments HHV-8 DNA was detected in 10 of 12 (83%) CD34 positive cell fractions. All cell subpopulations from the non-KS group were HHV-8 negative, with the exception of one positive B cell sample obtained from an HIV-infected patient. Our data demonstrate that in peripheral blood HHV-8 is detectable not only in CD19+ cells, as previously reported, but also in other cells, including T cells, monocytes, and cells devoid of specific lineage markers. We also show for the first time that CD34+ cells in peripheral blood of KS patients are a predominant HHV-8-harboring population, suggesting that they represent an additional important reservoir for this virus in vivo.

Induction of prostacyclin receptor expression in human erythroleukemia cells.

We have identified both high-affinity (KD = 36 +/- 3 nM) and low-affinity (KD = 2.1 +/- 0.8 microM) prostacyclin (PGI2)-receptor sites on human erythroleukemia (HEL) cells using the radiolabelled prostacyclin analogue. [3H]iloprost. The addition of the phorbol ester, TPA, to the culture medium caused a 5-10-fold increase in the number of both the low- and the high-affinity sites, without any change in their affinity constants. Iloprost stimulated HEL cell membrane adenylate cyclase activity 5-fold. This stimulation was potentiated in the presence of GTP, indicating a conventional PGI2 receptor-G2-adenylate cyclase system. HEL cells represent a source of prostacyclin receptor mRNA which may be of value in expression cloning of this receptor.

HIV-1-specific immunity in persistently seronegative individuals at high risk for HIV infection.

A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 4/94 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.

Human immunodeficiency virus type 1 glycoprotein 120-specific T lymphocytes provide intermolecular help for anti-CD4 autoantibody production in exposed uninfected subjects.

Anti-CD4 antibodies have been documented in about 10-20% of HIV-infected patients. This autoimmune response could be triggered by increased CD4 processing and unveiling of hidden (cryptic) epitopes. Multiple markers of exposure to HIV have been described in exposed uninfected individuals. Here, we investigated the mechanisms underlying the generation of anti-CD4 antibodies in a cohort of 54 seronegative exposed uninfected individuals. We identified anti-CD4 antibodies above normal levels in 16 of 47 (34%) exposed uninfected subjects. The fine specificity of these antibodies was different in this cohort when compared with those found in HIV+ patients. This suggested the possibility of different mechanisms underlying the generation of anti-CD4 antibodies in these two groups. Indeed, in exposed uninfected subjects, we found circulating CD4 T cells specific for gp120, but not for CD4. In contrast, HIV-1-seropositive patients had peripheral blood T cells specific for both molecules. Noncovalent binding of gp120 to soluble CD4 enhanced activation of gp120-specific T lymphocytes in exposed uninfected subjects, but not in HIV+ subjects. Moreover, gp120-specific T cells isolated from exposed uninfected, but not from HIV+, subjects provided help for anti-CD4 antibody production by B cells pulsed with CD4-gp120 complex. We conclude that gp120-specific T cells are present in exposed uninfected individuals, and can provide intermolecular help for anti-CD4 antibody production. This mechanism is distinct from that found in HIV-1-seropositive patients and may play a protective role against HIV-1 infection in vivo.

Heterogeneity in exposed uninfected individuals.

In spite of repeated exposures to HIV, some individuals remain seronegative and apparently uninfected. A variety of mechanisms potentially able to confer resistance to HIV infection, including cell-mediated and (unconventional) humoral immune responses, as well as mutations affecting receptors for virus entry have been considered and analysed. In this article, we want to discuss recent reports on specific immune responses and genetic factors potentially involved in mechanisms of protection, and to present some of our data relative to a cohort of people sexually exposed to HIV-1, but persistently seronegative. These EU (exposed uninfected) individuals can be distinguished from "normal" unexposed controls on the basis of significantly increased frequencies of a number of immunological parameters that might be considered "unconventional" correlates of HIV infection/protection. However, EU individuals are highly heterogeneous since the various unconventional immune responses considered can be present in all possible combinations. Aim of future research will be to ascertain the role of such immune responses in the maintenance of the protection state, or their secondary nature as signals of a particular kind of infection.

Increased plasma levels of the C-C chemokine RANTES in patients with primary HIV-1 infection.

To investigate the role played by chemokines in the natural history of human immunodeficiency virus (HIV) infection, we measured the plasma levels of RANTES. MIP-1 alpha and MIP-1 beta in a cohort of patients with primary HIV-1 infection (PHI) followed longitudinally. The cohort included 17 patients with well-documented history of acute HIV syndrome within two months of the first observation. The mean plasma concentration of RANTES, but not that of MIP-1 alpha or MIP-1 beta, was significantly higher in patients with PHI (192.3 ng/ml) than in five HIV-seronegative controls (8.0 ng/ml) studied during the same time period. Treatment of blood with a cocktail of drugs preventing platelet activation, followed by high-speed centrifugation, reduced the levels of RANTES by approximately 2 logs both in patients and in controls, indicating that the bulk of RANTES was released by platelets, which are known to store this chemokine in their alpha-granules, in the immediate aftermath of blood drawing. No correlation was seen between the levels of RANTES and the number of HIV genome equivalents in plasma. These data suggest that large amounts of pre-formed RANTES are stored in platelets and, possibly, in other blood cells during the early phases of HIV infection. The possible role of this HIV-suppressive chemokine in the control of viral replication during PHI remains to be established.

Prognostic factors and effectiveness of treatment in acute renal failure due to multiple myeloma: a review of 50 cases. Report of the Italien Renal Immunopathology Group.

We studied 50 patients with myeloma acute renal failure to investigate possible prognostic factors and to evaluate the effectiveness of the various treatment schedules used. Renal failure was reversible 1 month after the onset in 50% of the patients considered. The patients treated with chemotherapy and plasma exchange recovered renal function more frequently (61% of the cases) than those treated only with chemotherapy (27%). The most important clinical prognostic factors were total proteins, serum creatinine values and myeloma type. Considering the histological findings, the prognosis correlated with the severity of the lesions and number of tubular casts. Survival at 1 year was higher in the patients who regained renal function than in those in whom renal function did not improve.

Heterogeneity of prostaglandin H2/thromboxane A2 receptors: distinct subtypes mediate vascular smooth muscle contraction and platelet aggregation.

Studies of the hierarchies of agonist and antagonist affinity for the prostaglandin (PG)H2/thromboxane (Tx)A2 receptor have been performed to establish whether distinct receptor subtypes exist in platelets and vascular smooth muscle cells (VSMC). They have yielded conflicting results. The pattern of homologous desensitization of phospholipase C activation and [Ca++i] increase induced by the PGH2/TxA2 agonist U46619 in rat aortic SMC was similar to that previously observed in human platelets: rapid desensitization of both responses followed by a delayed loss of binding sites from the cell membrane. Recently, the pattern of receptor inactivation by the antagonist ligand, GR 32191, has identified two subtypes in platelets. GR 32191 binds reversibly (GRr) to a site that mediates platelet shape change and an increase [Ca++i] and irreversibly (GRirr) to a site linked to phospholipase C activation and aggregation. In contrast to platelets, studies of ligand dissociation only identified GRr sites in rat aortic SMC and GR 32191 failed to inactivate PGH2/TxA2 receptors as detected by the PGH2/TxA2 receptor antagonist, [3H]SQ 29548. Inhibition of U46619-induced contraction of both rat aortic and human saphenous vein was competitive, consistent with the absence of GRirr sites in VSMC. Platelet activating factor, which heterologously desensitizes U46619-evoked phospholipase C activation in platelets, had no such effect in VSMC. The biochemical events attendant to PGH2/TxA2 receptor desensitization are similar in SMC and platelets. However, both the pattern of receptor inactivation by GR 32191 and of heterologous desensitization by PAF, suggest that VSMC lack the receptor subtype that transduces aggregation of platelets.

Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.

Beta-1 integrins in the normal human glomerular capillary wall: an immunoelectron microscopy study.

The localization of the alpha 2, alpha 3 and alpha 6 subunits of the beta 1 integrin family on different cells of the glomerular capillary wall and on juxta-capillary mesangium was investigated using an immunoelectron microscopic technique on freshly harvested normal human glomeruli. Alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 1 were weakly expressed on both luminal and abluminal surfaces of glomerular endothelial cells; alpha 2 beta 1 and alpha 3 beta 1 were also found on the mesangium of the juxta-capillary areas. Alpha 3 beta 1 was regularly present in great density on the basal and lateral surface of podocyte foot processes, confirming alpha 3 beta 1 as the unique beta 1 integrin on glomerular epithelial cells. None of these integrins was strictly polarized along the glomerular basement membrane, thus suggesting, in agreement with recent literature, that these molecules perform other biological functions in addition to adhesivity.

Very late activation-3 integrin is the dominant beta 1-integrin on the glomerular capillary wall: an immunofluorescence study in nephrotic syndrome.

The expression of alpha 2; alpha 3; alpha 5; alpha 6-subunits of the beta 1 [very late activation (VLA)] integrin family was studied in kidney specimens using an immunofluorescent technique. 6 specimens from normal kidney were compared with 10 specimens from patients affected by various glomerulopathies [minimal change nephropathy (MCN), membranous nephropathy (MN) and systemic lupus erythematosus nephritis (SLEN)]. On normal glomeruli, alpha 3 was the dominant integrin, being mainly present on podocytes and showing a linear fluorescent pattern codistributed with laminin. In MCN and SLEN, alpha 3 presented a normal pattern. In MN, alpha 3 revealed a trabecular picture on thickened glomerular basement membranes. Moreover, in stage-III MN, a segmental loss of alpha 3-integrin was detected. In our opinion, VLA-3 may offer an interesting approach to the study of the relationships between podocytes and their substrate.

Cyclosporin-induced endothelial cell injury.

The administration of Cyclosporin-A (CyA) to animals and humans may induce an arteriolar damage. It has also been reported that CyA in some instances may cause an hemolytic uremic-like syndrome. This is a syndrome of vascular damage with thrombotic occlusions of the microcirculation. Endothelial injury is considered the first event in the pathogenetic cascade leading to hemolytic-uremic syndrome. We have used bovine aortic endothelial cells in culture to address the issue of CyA-induced arteriolar damage. Exposure of endothelial cells to different concentrations of CyA induced a time- and dose-dependent cell injury in vitro. The damage induced by CyA was characterized by an early cell detachment from culture substrate followed by cell lysis as documented by the increase in lactate dehydrogenase (LDH) and 51Cr release. Both detachment and lysis were negligible after short-term incubation of 1 microM CyA with endothelial cells. One micromolar CyA only induced lysis if incubations were prolonged above 6 hours. Ten and 50 microM CyA both induced marked endothelial cell detachment and lysis; lysis started 3 hours after incubation of endothelial cells with CyA and was maximal at the end of 24 hours incubation. CyA-induced injury was associated with dose- and time-dependent increase in prostacyclin and thromboxane A2 release by endothelial cells exposed to CyA independently from the concentrations of CyA used. CyA-induced generation of prostacyclin and thromboxane A2 was inhibited when the incubations were performed in the presence of aspirin (500 microM). These studies indicate that CyA exerts a direct cytotoxic effect on endothelial cells and might help in understanding the pathogenesis of CyA-induced vascular damage.

Point mutation in the seventh hydrophobic domain of the human thromboxane A2 receptor allows discrimination between agonist and antagonist binding sites.

Thromboxane A2, a potent platelet agonist and vasoconstrictor, exerts its actions via specific G protein-coupled receptors. cDNAs encoding the full length thromboxane receptor have been isolated from human placenta mRNA by reverse transcriptase-polymerase chain reaction. An expression construct, under control of the cytomegalovirus promoter, was introduced into human embryonic kidney 293 cells. Membranes from transfected cells bound the thromboxane antagonist SQ29,548 and the agonist [15-(1 alpha,2 beta(5z)-3 alpha(1E,3S)-4 alpha)]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid) with high affinities, and significantly more receptors were expressed in these cells, compared with platelet preparations. The putative seventh transmembrane segment is highly related in all cloned members of the eicosanoid receptor family and forms a critical portion of the ligand binding pocket for G protein-coupled receptors. Several point mutations in this segment were generated. Binding of SQ29,548 was virtually abolished in cells transfected with all the variant receptor constructs. However, one receptor variant (TxR-W299L), in which a tryptophan at position 299 was substituted for a leucine residue, allowed a definite discrimination between agonist and antagonist binding sites in competition and saturation binding experiments. An antibody directed toward the third intracellular loop of the thromboxane receptor was able to immunoprecipitate native thromboxane receptor in solubilized membranes from human erythroleukemia cells and transfected cells.

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse.

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-alpha-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.

Acute renal failure of medical type in an elderly population.

One hundred and nine unselected patients with Acute Renal Failure (ARF) of medical aetiology were hospitalized at the Nephrological Unit of Policlinico University Hospital (Modena) during a 30-month period. ARF was considered as a rapid increase of serum creatinine > 2mg/dl over the baseline level or the doubling of pre-existing value in chronic renal failure. Mean age of patients was 67+/-17 years and median age was 72; 64.2% needing dialytic treatment. Four main causes of ARF were identified: 33 patients had reduced renal perfusion by dehydration, hypotension etc.; 20 multifactorial aetiology; 14 biopsy-investigated renal parenchymal diseases and 39 had drug-related acute renal failure (D-ARF). The clinical outcome was significantly worse in elderly patients as regard mortality (P < 0.02), chronic dialytic treatment (P < 0.04) and complete recovery (P < 0.004). The mean age of D-ARF patients was significantly greater than remaining ARF patients (72.6+/-12.8 vs 63.2+/-18.5: P < 0.004. Nonsteroidal antiinflammatory drugs (NSAIDs) and ACE-inhibitors (Ace-i) caused ARF in 24 and 8 patients respectively. Elderly age, vascular disease and monoclonal gammopathy represented the main risk factors and were significantly more frequent in D-ARF patients (P<001, <0.01, <0.04 respectively). Our data confirm the high susceptibility of ageing kidneys to nephrotoxic damage caused by drugs affecting glomerular autoregulation by microvascular mechanisms. Greater attention to renal changes in ageing and an increased dissemination of preventative measures among nephrologists, could reduce the incidence of these serious and potentially lethal diseases.