Modulation of Gr1low monocyte subset impacts insulin sensitivity and weight gain upon high-fat diet in female mice.

BACKGROUND/OBJECTIVES: Blood monocytes are expanded during obesity. However, the differential contribution of monocyte subsets in obesity-related metabolic disorders remains unknown. The aim of the study was to define the role of the Gr1low monocyte subset upon high-fat diet (HFD). METHODS: We used transgenic female mouse models allowing the modulation of circulating Gr1low monocyte number (decreased number in CX3CR1-/- mice and increased number in CD11c-hBcl2 mice) and studied obesity upon HFD. RESULTS: We reported here that HFD induced monocytosis in mice, preferentially due to Gr1low monocyte expansion, and was associated with a specific upregulation of CD11c on that subset. Using mice models with altered Gr1low monocyte number, we found a striking correlation between Gr1low monocytes, bodyweight (BW) and insulin resistance (RT) status. Indeed, CX3CR1-/- female mice, with reduced Gr1low monocytes upon HFD, showed increased RT and a pro-inflammatory profile of the adipose tissue (AT) despite a lower BW. Conversely, mice expressing the anti-apoptotic gene hBcl2 in CD11c-expressing cells have increased Gr1low monocytes, higher insulin sensitivity upon HFD and an anti-inflammatory profile of the AT. Finally, increasing Gr1low monocytes in Gr1low-defective CX3CR1-/- mice rescued BW loss in these mice. CONCLUSIONS: By using transgenic female mice and adoptive transfer experiments, we established the evidence for a correlation between Gr1low monocyte subset and weight gain and RT. Hence, this specific Gr1low monocyte subset could be used as a target for acting on AT inflammation and RT.

Modelling Ebola within a community.

The 2014 Ebola epidemic was the largest on record. It evidenced the need for improved models of the spread of Ebola. In this research we focus on modelling Ebola within a small village or community. Specifically, we investigate the potential of basic Susceptible-Exposed-Infectious-Recovered (SEIR) models to describe the initial Ebola outbreak, which occurred in Meliandou, Guinea. Data from the World Health Organization is used to compare the accuracy of various models in order to select the most accurate models of transmission and disease-induced responses. Our results suggest that (i) density-dependent transmission and mortality-induced behavioural changes shaped the course of the Ebola epidemic in Meliandou, while (ii) frequency-dependent transmission, disease-induced emigration, and infection-induced behavioural changes are not consistent with the data from this epidemic.

Physiopathology of necrobiotic xanthogranuloma with monoclonal gammopathy.

RATIONALE: Xanthomatosis associated with monoclonal gammopathy includes hyperlipidaemic xanthoma (HX), normolipidaemic xanthoma (NX) and necrobiotic xanthogranuloma (NXG). All three pathologies are characterized by skin or visceral lesions related to cholesterol accumulation, monoclonal immunoglobulin (MIg) and hypocomplementemia. The pathophysiology underlying NXG remains unknown although the involvement of MIg is suspected. OBJECTIVE: To provide further insights into the pathophysiology of NXG, we evaluated the plasma lipid phenotype, mechanisms involved in cellular cholesterol accumulation and role of MIg in an analysis of blood and plasma markers of inflammation in 16 patients with xanthomatosis [NXG (n = 8) and NX (n = 8)] associated with monoclonal IgG relative to the relevant controls. RESULTS: The lipid profile of patients with NXG was characterized by a low HDL-C phenotype and an abnormal distribution of HDL particles. Sera from patients with NXG induced cholesterol accumulation in human macrophages. This accumulation was due in part to a significant reduction in the HDL capacity to promote cholesterol efflux from macrophages, which was not found in the case of NX. The MIg of NXG and NX patients was tested positively by ELISA to recognize a large spectrum of lipoproteins. High plasma levels of pro-inflammatory cytokines (TNFα and IL-6), soluble cytokine receptors (sIL-6R, sTNFRI and sTNFRII), adhesion molecules (VCAM-1 and ICAM-1) and chemokines (MCP-1, IL-8 and MIP-1α) were observed in both patients with NXG and NX, revealing a specific xanthoma inflammatory signature which was inversely correlated with plasma levels of anti-inflammatory HDL. However, patients with NXG were distinguished by elevated levels of IL-15 and a marked increase in the rate of intermediate CD14++CD16+ monocytes. CONCLUSION: This study revealed that NXG is characterized by impaired macrophage lipid homeostasis associated with a systemic inflammatory profile that may result from the interaction of MIg and lipoproteins.

Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study.

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cell-rich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

Early activation of the cardiac CX3CL1/CX3CR1 axis delays ß-adrenergic-induced heart failure.

We recently highlighted a novel potential protective paracrine role of cardiac myeloid CD11b/c cells improving resistance of adult hypertrophied cardiomyocytes to oxidative stress and potentially delaying evolution towards heart failure (HF) in response to early ß-adrenergic stimulation. Here we characterized macrophages (Mφ) in hearts early infused with isoproterenol as compared to control and failing hearts and evaluated the role of upregulated CX3CL1 in cardiac remodeling. Flow cytometry, immunohistology and Mφ-depletion experiments evidenced a transient increase in Mφ number in isoproterenol-infused hearts, proportional to early concentric hypertrophy (ECH) remodeling and limiting HF. Combining transcriptomic and secretomic approaches we characterized Mφ-enriched CD45+ cells from ECH hearts as CX3CL1- and TNFα-secreting cells. In-vivo experiments, using intramyocardial injection in ECH hearts of either Cx3cl1 or Cx3cr1 siRNA, or Cx3cr1-/- knockout mice, identified the CX3CL1/CX3CR1 axis as a protective pathway delaying transition to HF. In-vitro results showed that CX3CL1 not only enhanced ECH Mφ proliferation and expansion but also supported adult cardiomyocyte hypertrophy via a synergistic action with TNFα. Our data underscore the in-vivo transient protective role of the CX3CL1/CX3CR1 axis in ECH remodeling and suggest the participation of CX3CL1-secreting Mφ and their crosstalk with CX3CR1-expressing cardiomyocytes to delay HF.

Attention-related increases in cortical responsivity dissociated from the contingent negative variation.

Certain tasks which increase attention to stimuli also elicit the contingent negative variation and increase the amplitude of the P300 component of the sensory evoked response. Therefore it appeared possible that the contingent negative variation and attention-related increases in P300 are either confounded by artifact or generated by common neural mechanisms. The fact that we have recorded attention-related increases in P300 amplitude independent of corresponding systematic changes in contingent negative variation indicates that neither of these possibilities is correct. The two phenomena are independently variable modulations of cortical activity.

Auditory evoked potentials during speech perception.

Neural responses evoked by the same binaural speech signal were recorded from ten right-handed subjects during two auditory identification tasks. One task required analysis of acoustic parameters important for making a linguistic distinction, while the other task required analysis of an acoustic parameter which provides no linguistic information at the phoneme level. In the time interval between stimulus onset and the subjects' identification responses, evoked potentials from the two tasks were significantly different over the left hemisphere but identical over the right hemisphere. These results indicate that different neural events occur in the left hemisphere during analysis of linguistic versus nonlinguistic parameters of the same acoustic signal.

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells.

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites.

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.

Neurologic, neuropsychologic, and computed cranial tomography scan abnormalities in 2- to 10-year survivors of small-cell lung cancer.

In order to evaluate the relationship between neurologic function and cranial irradiation, 20 patients treated on National Cancer Institute (NCI) small-cell lung cancer (SCLC) trials who were alive and free of cancer 2.4 to 10.6 years (median, 6.2) from the start of therapy were studied. All were tested with a neurologic history and examination, mental status examination, neuropsychologic testing, and review of serial computed cranial tomography (CCT) scans. Fifteen patients had been treated with prophylactic cranial irradiation (PCI), two patients with therapeutic cranial irradiation, and three received no cranial irradiation. All patients but one were ambulatory and none were institutionalized. Fifteen patients (75%) had neurologic complaints, 13 (65%) had abnormal neurologic examinations, 12 (60%) had abnormal mental status examinations, 13 (65%) had abnormal neuropsychologic testing, and 15 (75%) had abnormal CCT scans. Compared with those given low-dose maintenance chemotherapy during PCI using 200 to 300 rad per fraction, patients who were given high-dose induction chemotherapy during the time of cranial irradiation or large radiotherapy fractions (400 rad) were more likely to have abnormal mental status examinations (6/6 v 4/9) and abnormal neuropsychologic tests (6/6 v 4/9), but no major difference in CCT findings was present. CCT scans in the majority of cases (11/18) showed progressive ventricular dilatation or cerebral atrophy up to 8 years after stopping therapy. We conclude neurologic abnormalities are common in long-term survivors of SCLC, and may be more prominent in patients given high-dose chemotherapy during cranial irradiation or treated with large radiotherapy fractions. The CCT scan abnormalities are common and progressive years after prophylactic cranial irradiation and chemotherapy are stopped.

The role of IL-10 in iNOS and cytokine mRNA expression during in vitro differentiation of bovine mononuclear phagocytes.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

A flow cytometric method for assessing viability of intraerythrocytic hemoparasites.

We have developed a rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites. The assay involves the selective uptake and metabolic conversion of hydroethidine to ethidium by live parasites present in intact erythrocytes. The red fluorescence imparted by ethidium intercalated into the DNA of the parasite permits the use of flow cytometry to distinguish infected erythrocytes with viable parasites from uninfected erythrocytes and erythrocytes containing dead parasites. Comparison of the fluorochromasia technique of enumerating the number and viability of hemoparasites in cultured erythrocytes with enumeration in Giemsa-stained films and uptake of [3H]hypoxanthine demonstrated the fluorochromasia technique yields comparable results. Studies with the hemoparasite, Babesia bovis, have shown the fluorochromasia technique can also be used to monitor the effect of parasiticidal drugs on parasites in vitro. The cumulative studies with the fluorochromasia assay suggest the assay will also prove useful in investigations focused on analysis of the immune response to hemoparasites and growth in vitro.

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites.

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.

Babesia bovis immunity. In vitro and in vivo evidence for IL-10 regulation of IFN-gamma and iNOS.

IL-10 has been shown to have profound immunoregulatory attributes and in the bovine appears to downregulate both Th1- and Th2-like responses. Using RT-PCR, we demonstrate IL-10 in vitro down-regulation of mRNA expression of iNOS, the cytokines involved in nitric oxide signal transduction initiation (IFN-gamma and TNF-alpha), and other mononuclear phagocyte associate cytokines. In addition, using RT-PCR with peripheral blood leukocytes and spleen leukocytes, the Griess reaction, and a killing assay, we provide evidence for the importance of iNOS in a successful immune response to B. bovis infection and for high and persistent IL-10 mRNA expression when the immune response is unsuccessful. We also provide evidence that antibody developed early after an initial infection appears to lack protective attributes (neutralizing and opsonic). Together, the data suggests that IL-10 and IFN-gamma are critical molecules involved in the response to this intraerythrocytic protozoan infection.

Bovine babesiosis. Seroprevalence and ticks associated with cattle from two different regions of Morocco.

A total of 475 bovine sera collected in 1995-1996 from 10 areas belonging to two different bioclimatic strata were tested for antibody activity to Babesia bigemina and Babesia bovis using indirect immunofluorescence (IIF). In the Gharb, the B. bovis seroprevalence was 21.7% and for B. bigemina, 10.8%. The infection rate for either or both species as determined microscopically with Giemsa-stained blood films was 18.9%. The Tiflet area was considered an endemic focus, and the seroprevalence was 42.2% for B. bovis and 40% for B. bigemina. The infection rate by stained blood film microscopy was 66.6%. In the Haouz region, only B. bovis was found, and the seroprevalence was 10.1% with 9.4% microscopically positive blood films. More than 80% of the cattle surveyed were infested by ticks and the mean infestation rate was 36 ticks per animal and 21 ticks per animal in the Gharb and Haouz, respectively. Six species were identified. Hyalomma marginatum, Hyalomma detritum, Hyalomma anatolicum Rhipicephalus bursa, Rhipicephalus sanguineous and Boophilus annulatus. Boophilus annulatus was found in both regions with high prevalence in the Gharb (31.3%). No further correlation was made between the identified species as vectors and the presence of B. bovis and B. bigemina in these areas.

Reactive oxygen and nitrogen intermediates and products from polyamine degradation are Babesiacidal in vitro.

Products released from activated macrophages have been demonstrated to have microbicidal activity against a variety of microorganisms. Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) have been shown to affect the induction of degenerate (crisis) forms of Plasmodium spp. Polyamines are degraded into acrolein which has also been shown to be toxic to Plasmodium spp. We have investigated the possibility that these products act similarly with Babesia bovis. Crisis forms of B. bovis developed in erythrocyte cultures after the introduction of supernatants containing ROI, RNI, and acrolein. Xanthine degradation by xanthine oxidase leads to the formation of superoxide anion, hydrogen peroxide, and hydroxyl radicals. The degradation in the presence of B. bovis was toxic to the parasite. The toxicity was partially reversed by the addition of the ROI scavenger catalase. However, H2O2 added directly had little effect, suggesting a role for the other ROI products. Spermine degradation by polyamine oxidase and direct addition of acrolein was toxic in a dose-dependent manner. Finally, spontaneous generation of nitric oxide from sodium nitroprusside or S-nitroso-N-acetyl-penicillamine was also toxic in a dose-dependent manner. These data lead us to suggest a role for activated macrophages in the primary immune response against B. bovis.

Age-related innate immune response in calves to Babesia bovis involves IL-12 induction and IL-10 modulation.

There is a strong innate immunity in calves to infection with Babesia bovis. Interleukin (IL)-12 and IL-10 have been shown in vitro to be important immunoregulatory cytokines. Here we demonstrate in vivo that the protective innate response in young calves to infection with virulent B. bovis involves the early appearance of IL-12 and interferon-gamma (IFN-gamma) transcripts in the spleen. In contrast, IL-12 and IFN-gamma mRNA expression in the spleens of adult cattle that succumbed to the infection was delayed and depressed and occurred within the context of IL-10 expression. Also in contrast with calves, there was no detectable antibody response before death in adults. A vigorous CD8+ T-cell expansion occurred in the spleens of both calves and adults.

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

Accuracy of ultrasound in the diagnosis of acute appendicitis compared with the surgeon's clinical impression.

OBJECTIVE: To compare the accuracy of the surgeon's clinical diagnosis of acute appendicitis with that of an ultrasonographic examination of the abdomen. DESIGN: Prospective trial. SETTING: US Navel Hospital, San Diego, Calif. PATIENTS: One hundred ten patients admitted to the hospital with suspected appendicitis from May 1990 to June 1992. INTERVENTION: Symptoms and signs for each patient were recorded, along with the surgeon's clinical impression of immediate surgery or observation. The patient then underwent an ultrasound examination performed by a staff radiologist. On the basis of the ultrasound findings the patient was placed into one of three categories: appendicitis, normal examination results, or other conditions. Patients with an ultrasound-based diagnosis of appendicitis proceeded to the operation, regardless of the surgeon's clinical impression. Those with other conditions diagnosed with ultrasonography were treated as was appropriate for the condition. RESULTS: The ultrasound-derived diagnosis of appendicitis had a sensitivity of 85.5%, a specificity of 84.4%, a positive predictive value of 88.3%, a negative predictive value of 80.1%, and an overall accuracy of 85.0%. The surgeon's clinical impression at the time of admission had a sensitivity of 62.9%, a specificity of 82.2%, a positive predictive value of 82.9%, a negative predictive value of 61.7%, and an overall accuracy of 71.2%. CONCLUSION: The overall accuracy of ultrasonography in the diagnosis of appendicitis was statistically superior to that of the surgeon's clinical impression (P < .0001). However, 24% of the patients with normal ultrasound findings were ultimately found to have appendicitis at operation, emphasizing the point that ultrasonography cannot be relied on to the exclusion of the surgeon's careful and repeated evaluation.