Local aromatase excess with recruitment of unusual promoters of CYP19A1 gene in prepubertal patients with gynecomastia.

OBJECTIVES: Gynecomastia may be due to aromatase excess in several diseases such as obesity and cancer. Aromatase excess syndrome (AEXS) is an autosomal dominant disorder caused by overexpression of CYP19A1. Germinal mutations occurring in AEXS include various genomic rearrangements including duplication, deletion, and inversion identified in the upstream region of CYP19A1. Aromatase overexpression caused by a CYP19A1 somatic mutation has been rarely described. METHODS: Breast adipose tissue biopsies or surgical specimens were obtained from 19 subjects with gynecomastia. Aromatase quantification was performed by digital PCR and CYP19A1 sequencing by RACE PCR products. RESULTS: We observed localized aromatase overexpression (>10 fold greater than normal) in breast adipose tissue from three prepubertal males with gynecomastia out of the 19 cases. One carried a chromosomal rearrangement between CYP19A1 and DMXL2, consistent with AEXS. In the 2 others, the first exon of CYP19A1 contained 11 different tissue-specific promoter subtypes, specifically I.4 or I.3 normally expressed by adipose tissue, but also the placental I.2 promoter and the more ubiquitous I.7 which is usually expressed in breast cancer, uterine, and endothelial tissues. No differences in clinical or biochemical characteristics were observed between these 3 subjects and 16 others without aromatase overexpression. CONCLUSIONS: We describe two cases of aromatase overexpression in breast adipose tissue associated with nonspecific promoter recruitment. Further investigations are necessary to understand the mechanisms involved in aberrant promoter selection.

Outcomes following prenatal diagnosis of isolated persistent left superior vena cava.

BACKGROUND: Prenatal diagnosis of persistent left superior vena cava is increasing, but little is known about outcomes of infants with prenatally diagnosed isolated persistent left superior vena cava. OBJECTIVE: To assess the outcomes of infants with isolated persistent left superior vena cava diagnosed prenatally compared with infants with associated malformations. METHODS: All cases of persistent left superior vena cava confirmed by specialized fetal echocardiography in pregnant women were included from a single-centre prospective registry. Unfavourable outcome was defined as termination of pregnancy, in utero death, postnatal death or severe genetic syndrome missed prenatally. RESULTS: A total of 256 infants were included: 113 cases (44.1%) with isolated persistent left superior vena cava and 143 cases (55.9%) with associated malformations; respectively, 111 (98.2%) and 101 (70.6%) had a live birth. The median postnatal clinical follow-up was 3.6 years. Five-year postnatal survival with good outcome was estimated at: 100% (95% confidence interval 90.7% to 100%) in infants with isolated persistent left superior vena cava; 91.0% (74.0% to 98.1%) in infants with associated cardiac anomalies; 87.5% (51.8% to 97.3%) in infants with associated extracardiac anomalies; 81.0% (52.6 to 94.6%) in infants with both cardiac and extracardiac anomalies; and 78.9% (36.7% to 95.9%) in infants with non-structural anomalies. All genetic findings and syndromes were detected in fetuses or infants with non-isolated persistent left superior vena cava. CONCLUSION: Infants with isolated persistent left superior vena cava have good short-term outcomes postnatally, but persistent left superior vena cava is frequently associated with other malformations that have an effect on outcomes, which should be thoroughly searched for prenatally.

A NEW COCCIDIAN (APICOMPLEXA: EIMERIIDAE) IN THE CRITICALLY ENDANGERED CENTRAL AMERICAN RIVER TURTLE (DERMATEMYS MAWII) IN BELIZE.

As part of a biannual health examination, coprological samples from 3-mo-old Central American river turtles, Dermatemys mawii (Gray, 1847) in a breeding program in Belize, Central America, revealed a previously undescribed coccidian (Apicomplexa) in 17 of 46 (37%) samples. Of 3 positive fecal samples transported to the University of Florida, coccidian oocysts were observed in 1 sample. Sporulated oocysts were measured and described, and using polymerase chain reaction (PCR), an approximately 400-base pair (bp) region of both the small subunit (18S) ribosomal RNA gene and 1,200-bp region of the internal transcribed spacer (ITS) gene were amplified in all 3 samples and their products were sequenced. For comparative value, the same PCR reactions and amplifications were performed on a fecal sample containing oocysts of Eimeria mitraria obtained from a red-eared slider, Trachemys scripta elegans. Results indicated a new eimerian in D. mawii, Eimeria grayi n. sp.

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

A key process of the nonstationary relationship between ENSO and the Western Pacific teleconnection pattern.

Recent studies have discovered an intriguing nonstationary relationship between El Ninõ-Southern Oscillation (ENSO) and the Western Pacific (WP) teleconnection pattern, one of the most prominent winter atmospheric circulation patterns in the North Pacific, with a regime-dependent interdecadal modulation of significant and insignificant correlations. However, the physical process underlying the observed nonstationary ENSO-WP relationship is a puzzle and remains to be elucidated, which is also essential for clarifying the still-debated nontrivial issue on whether the WP is directly forced by ENSO or by midlatitude storm tracks-driven intrinsic processes. Based on empirical orthogonal function (EOF) analysis of the upper-tropospheric teleconnection patterns and associated Rossby wave sources (RWS), we show that the nonstationarity in question is due to the regime-dependent constructive or destructive interference in meridional overturning circulation between the two leading EOFs of RWS best correlated with ENSO and WP, respectively. The observed insignificant correlation between ENSO and the WP after the 1988 regime shift can be explained by interrupted teleconnection between the tropics and high latitudes due to the collapse of the subtropical bridge pillar in the jet entrance region, consequence of the destructive interference. This suggested interference mechanism related to the regime-dependent upper-level RWS fields has significant implications for resolving the puzzle that hinders better understanding of decadal regime behaviors of the climate system in the North Pacific.

Autocrine Adenosine Regulates Tumor Polyfunctional CD73+CD4+ Effector T Cells Devoid of Immune Checkpoints.

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4+ T cells (Teffs) enriched in polyfunctional Th1.17 cells characterized by expression of CXCR3, CCR6, and MDR1, and production of IL17A/IFNγ/IL22/GM-CSF. CD39+ Tregs selectively targeted CD73+ Teffs through cooperative degradation of ATP into Ado inhibiting and restricting the ability of CD73+ Teffs to secrete IL17A. CD73+ Teffs infiltrating breast and ovarian tumors were functionally blunted by Tregs expressing upregulated levels of CD39 and ATPase activity. Moreover, tumor-infiltrating CD73+ Teffs failed to express inhibitory immune checkpoints, suggesting that CD73 might be selected under pressure from immune checkpoint blockade therapy and thus may represent a nonredundant target for restoring antitumor immunity.Significance: Polyfunctional CD73+ T-cell effectors lacking other immune checkpoints are selectively targeted by CD39 overexpressing Tregs that dominate the breast tumor environment. Cancer Res; 78(13); 3604-18. ©2018 AACR.

A novel regulation of PD-1 ligands on mesenchymal stromal cells through MMP-mediated proteolytic cleavage.

Whether fibroblasts regulate immune response is a crucial issue in the modulation of inflammatory responses. Herein, we demonstrate that foreskin fibroblasts (FFs) potently inhibit CD3+ T cell proliferation through a mechanism involving early apoptosis of activated T cells. Using blocking antibodies, we demonstrate that the inhibition of T cell proliferation occurs through cell-to-cell interactions implicating PD-1 receptor expressed on T cells and its ligands, PD-L1 and PD-L2, on fibroblasts. Dual PD-1 ligand neutralization is required to abrogate (i) binding of the PD-1-Fc fusion protein, (ii) early apoptosis of T cells, and (iii) inhibition of T cell proliferation. Of utmost importance, we provide the first evidence that PD-1 ligand expression is regulated through proteolytic cleavage by endogenous matrix metalloproteinases (MMPs) without transcriptional alteration during culture-time. Using (i) different purified enzymatic activities, (ii) MMP-specific inhibitors, and (iii) recombinant human MMP-9 and MMP-13, we demonstrated that in contrast to CD80/CD86, PD-L1 was selectively cleaved by MMP-13, while PD-L2 was sensitive to broader MMP activities. Their cleavage by exogenous MMP-9 and MMP-13 with loss of PD-1 binding domain resulted in the reversion of apoptotic signals on mitogen-activated CD3+ T cells. We suggest that MMP-dependent cleavage of PD-1 ligands on fibroblasts may limit their immunosuppressive capacity and thus contribute to the exacerbation of inflammation in tissues. In contrast, carcinoma-associated fibroblasts appear PD-1 ligand-depleted through MMP activity that may impair physical deletion of exhausted defective memory T cells through apoptosis and facilitate their regulatory functions. These observations should be considered when using the powerful PD-1/PD-L1 blocking immunotherapies.

Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance.

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells.

Human natural killer cells promote cross-presentation of tumor cell-derived antigens by dendritic cells.

Dendritic cells (DCs) cross-present antigen (Ag) to initiate T-cell immunity against most infections and tumors. Natural killer (NK) cells are innate cytolytic lymphocytes that have emerged as key modulators of multiple DC functions. Here, we show that human NK cells promote cross-presentation of tumor cell-derived Ag by DC leading to Ag-specific CD8(+) T-cell activation. Surprisingly, cytotoxic function of NK cells was not required. Instead, we highlight a critical and nonredundant role for IFN-γ and TNF-α production by NK cells to enhance cross-presentation by DC using two different Ag models. Importantly, we observed that NK cells promote cell-associated Ag cross-presentation selectively by monocytes-derived DC (Mo-DC) and CD34-derived CD11b(neg) CD141(high) DC subsets but not by myeloid CD11b(+) DC. Moreover, we demonstrate that triggering NK cell activation by monoclonal antibodies (mAbs)-coated tumor cells leads to efficient DC cross-presentation, supporting the concept that NK cells can contribute to therapeutic mAbs efficiency by inducing downstream adaptive immunity. Taken together, our findings point toward a novel role of human NK cells bridging innate and adaptive immunity through selective induction of cell-associated Ag cross-presentation by CD141(high) DC, a process that could be exploited to better harness Ag-specific cellular immunity in immunotherapy.

Aberrant drainage of the umbilical vein into the coronary sinus without ductus venosus agenesis.

We describe a case series of 4 fetuses with ectopic connections of the ductus venosus to the coronary sinus detected prospectively between August 2011 and February 2012 in 2 congenital cardiologic centers. An enlarged coronary sinus alerted the sonographer. Fetal echocardiography showed ectopic connection of the ductus venosus in an enlarged coronary sinus in all 4 cases. To our knowledge, this anatomic form of ectopic umbilical vein drainage has not previously been reported. The infants were doing well. This venous variant should be considered in cases of isolated coronary sinus dilatation after elimination of a left superior vena cava and a totally anomalous pulmonary vein connection.

Tumor promotion by intratumoral plasmacytoid dendritic cells is reversed by TLR7 ligand treatment.

Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

ICOS-ligand expression on plasmacytoid dendritic cells supports breast cancer progression by promoting the accumulation of immunosuppressive CD4+ T cells.

Human breast tumors are infiltrated by memory CD4(+) T cells along with increased numbers of regulatory T cells (Treg) and plasmacytoid dendritic cells (pDC) that facilitate immune escape and correlate with poor prognosis. Here, we report that inducible costimulatory molecule (ICOS), a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, is expressed mostly by tumor-associated Treg in primary breast tumors. A large proportion of these ICOS(+) Treg were Ki67(+) and this evident proliferative expansion was found to rely on interactions with tumor-associated pDC. Indeed, tumor-associated Treg highly expanded in presence of pDC but failed to proliferate under CD3/CD28 signal. In vitro experiments revealed that the addition of a neutralizing anti-ICOS antibody blocked pDC-induced Treg expansion and interleukin-10 secretion by memory CD4(+) T cells, establishing a pivotal role for ICOS in this process. Supporting these findings, the presence of ICOS(+) cells in clinical specimens of breast cancer correlated with a poor prognosis. Together, our results highlight an important relationship between Treg and pDC in breast tumors, and show that ICOS/ICOS-L interaction is a central event in immunosuppression of tumor-associated memory CD4(+) T cells. These findings strongly rationalize antibody-mediated ICOS blockade as a powerful clinical strategy to correct immune escape and promote therapeutic responses in breast cancer.

Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.

Infiltration and dysfunction of immune cells have been documented in many types of cancers. We previously reported that plasmacytoid dendritic cells (pDC) within primary breast tumors correlate with an unfavorable prognosis for patients. The role of pDC in cancer remains unclear but they have been shown to mediate immune tolerance in other pathophysiologic contexts. We postulated that pDC may interfere with antitumor immune response and favor tolerance in breast cancer. The present study was designed to decipher the mechanistic basis for the deleterious impact of pDC on the clinical outcome. Using fresh human breast tumor biopsies (N = 60 patients), we observed through multiparametric flow cytometry increased tumor-associated (TA) pDC (TApDC) rates in aggressive breast tumors, i.e., those with high mitotic index and the so-called triple-negative breast tumors (TNBT). Furthermore, TApDC expressed a partially activated phenotype and produced very low amounts of IFN-α following toll-like receptor activation in vitro compared with patients' blood pDC. Within breast tumors, TApDC colocalized and strongly correlated with TA regulatory T cells (TATreg), especially in TNBT. Of most importance, the selective suppression of IFN-α production endowed TApDC with the unique capacity to sustain FoxP3(+) Treg expansion, a capacity that was reverted by the addition of exogenous IFN-α. These findings indicate that IFN-α-deficient TApDC accumulating in aggressive tumors are involved in the expansion of TATreg in vivo, contributing to tumor immune tolerance and poor clinical outcome. Thus, targeting pDC to restore their IFN-α production may represent an attractive therapeutic strategy to overcome immune tolerance in breast cancer.

Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1ß], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1ß and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1ß and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.

Quantitative and functional alterations of plasmacytoid dendritic cells contribute to immune tolerance in ovarian cancer.

In ovarian cancer, the immune system fails to eradicate established tumors partly due to the induction of immune tolerance within tumor microenvironment. In this study, we investigated the contribution of plasmacytoid dendritic cells (pDC) in the establishment of immune tolerance in a cohort of 44 ovarian cancer patients. In the tumor and malignant ascites, CD4(+)CD123(+)BDCA2(+) pDC were the most abundant dendritic cell subset; however, they were profoundly depleted in peripheral blood. The presence of pDC in primary ovarian cancer, but not ascites, was an independent prognostic factor associated with early relapse. Following chemotherapy, we observed a partial restoration of blood pDC levels in patients in complete remission. These findings show preferential recruitment of pDC into tumors where they express a partially mature phenotype that may reflect an in situ activation. Importantly, compared with pDC found in ascites or blood, tumor-associated pDC (TApDC) produced less IFN-α, TNF-α, IL-6, macrophage inflammatory protein-1ß, and RANTES in response to toll-like receptor stimulation, and alterations in pDC functions were mainly mediated through tumor-derived TNF-α and TGF-ß. Unlike ascites-derived pDC, TApDC induced IL-10 production from allogeneic naive CD4(+) T lymphocytes, suggesting the existence of a paracrine immunosuppressive loop. Taken together, our findings indicate that both local and systemic dysfunction of pDC play a critical role in the progression of ovarian cancer via induction of immune tolerance.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-gamma. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-gamma production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-gamma production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-gamma production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I-specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome.

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3(+) T cells are typical Treg based on their CD4(+)CD25(high)CD127(low)FOXP3(+) phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP(+) dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3(+), and CD8(+) T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem.

Prevalence of Toxocara spp., Toxascaris leonina and ancylostomidae in public parks and beaches in different climate zones of Costa Rica.

This epidemiological study was conducted in different regions of Costa Rica to determine the prevalence of the developmental stages of potential zoonotic intestinal helminths of dogs and cats in public places. Samples were collected within three main climate zones including rural and urban areas during both the rainy and the dry season. Faecal and environmental samples were taken from 69 parks and beaches. Of the faecal samples 3% contained Toxascaris spp. eggs, 7% Toxocara spp. eggs and 55% contained ancylostomidae eggs. Of the soil samples, 2% contained ancylostomidae eggs and 0.8% contained ascarid eggs. Significant differences in the presence of parasites were found in faecal samples of dry, moist and wet climate zones and between the dry and rainy seasons. Significant differences in the presence of eggs and larvae were also found in the grass samples in the dry, the moist and the wet climate zones and between the different seasons. No significant differences were found between rural and urban areas.

Effect of natural iron fertilization on carbon sequestration in the Southern Ocean.

The availability of iron limits primary productivity and the associated uptake of carbon over large areas of the ocean. Iron thus plays an important role in the carbon cycle, and changes in its supply to the surface ocean may have had a significant effect on atmospheric carbon dioxide concentrations over glacial-interglacial cycles. To date, the role of iron in carbon cycling has largely been assessed using short-term iron-addition experiments. It is difficult, however, to reliably assess the magnitude of carbon export to the ocean interior using such methods, and the short observational periods preclude extrapolation of the results to longer timescales. Here we report observations of a phytoplankton bloom induced by natural iron fertilization--an approach that offers the opportunity to overcome some of the limitations of short-term experiments. We found that a large phytoplankton bloom over the Kerguelen plateau in the Southern Ocean was sustained by the supply of iron and major nutrients to surface waters from iron-rich deep water below. The efficiency of fertilization, defined as the ratio of the carbon export to the amount of iron supplied, was at least ten times higher than previous estimates from short-term blooms induced by iron-addition experiments. This result sheds new light on the effect of long-term fertilization by iron and macronutrients on carbon sequestration, suggesting that changes in iron supply from below--as invoked in some palaeoclimatic and future climate change scenarios--may have a more significant effect on atmospheric carbon dioxide concentrations than previously thought.

Molecular characterization of Clostridium difficile clinical isolates in a geriatric hospital.

The discriminatory potential of a combination of various typing methods was evaluated on a set of 21 Clostridium difficile isolates obtained from symptomatic patients hospitalized in a geriatric unit and 7 non-toxigenic isolates from the same hospital. Isolates were firstly serotyped and toxinotyped. Of the 28 isolates, 19 belonged to serogroup A. PCR-ribotyping and PCR-RFLP on the fliC and slpA genes were then applied to these 19 isolates. The results suggest that the combination of PCR-ribotyping with PCR-RFLP analysis of slpA could be more discriminatory and suitable for studying C. difficile epidemiology.