Blood Feeding and Plasmodium Infection Alters the miRNome of Anopheles stephensi

Blood feeding is an integral process required for physiological functions and propagation of the malaria vector Anopheles. During blood feeding, presence of the malaria parasite, Plasmodium in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in Anopheles stephensi. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in An. stephensi. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level.     

Blood protein synthesis in pupae of the silkmoth, Hyalophora cecropia

The blood proteins of pupae of Hyalophora cecropia were studied by acrylamide gel electrophoresis. Analyses of fractions separated on Sephadex G-75 columns revealed that a series of low molecular weight proteins previously reported to be absent in diapausing pupae are a normal component of diapausing pupal blood. Incorporation of isotopically labelled leucine into blood protein bands of injured pupae, perfused pupae, and perfused isolated pupal abdomens (lacking a midgut) revealed that each of these three preparations could synthesize all of the low molecular weight protein bands, with maximal incorporation in the perfused whole pupae.     

Separation and labeling of specific subpopulations of Botryllus blood cells

Blood cells of the colonial tunicate Botryllus were separated by density gradient centrifugation in Percoll. Unseparated blood cells were used to immunize mice for development of hybridoma cell lines producing anti-Botryllus monoclonal antibodies. These antibodies identify specific subpopulations of blood cells, indicating possible divisions of these cells into defined subgroups sharing particular differentiation antigens. Additional studies utilizing fluorescein- conjugated lectins also revealed differential binding to density- and monoclonal antibody-defined blood cell fractions. These methods allow separation of the different Botryllus blood cell types for functional studies.     

Innervation of the microcirculatory bed of the human trigeminal nerve]

The investigation of film preparations and histological sections of human trigeminal nerves impregnated with silver nitrate and treated after Gomori, Falck--Hillarp demonstrated a rich innervation in the intraneural blood vessels. The most various and complex interconnections of the neural structures were noted in arterioles and venules of the node capsule, epineurium and external layers of perineurium of the trigeminal nerve branches. On the vessel walls of these layers, neural plexus were revealed. Sensitive innervation of the neural blood vessels mainly performed by posvalent tissue-vascular receptors. In the walls of intraneural vessels, adrenergic and cholinergic neural plexus are revealed.     

A noninvasive approach to studying fetal cells for prenatal diagnosis of chromosomal aneuploidies

Fetal cells isolated from maternal peripheral blood during the second trimester of pregnancy were analyzed. Blood samples were centrifuged in a Ficoll-Paque gradient, the mononuclear cell fraction was isolated and stained with fluorescent monoclonal antibodies against glycophorine A (GPA + PE), transferrin (CD71 + FITC), and Hoechst 33342. Fluorescence-activated cell sorting (FACS) was conducted on a Vantage flow cytofluorimeter (Becton Dickinson). Fluorescence in situ hybridization (FISH) with Y chromosome-specific DNA probe revealed fetal cells that exhibited Y signal in all 20 blood samples obtained from women pregnant with healthy male fetuses. The concentration of these fetal cells averaged about 1.34% and ranged from 0.1 to 4.2% in different blood samples. In six cases, blood samples were obtained from pregnant women, in which prenatal cytogenetic analysis revealed various fetal aneuploidies. Using FISH with DNA probes specific for chromosomes X, 18, and 13/21, Fetal cells with chromosomal aberrations were detected in these six maternal blood samples at a concentration from 1.5 to 5.6% (on average 3.7%). These results indicate the possibility of a new noninvasive approach, which is safe for both mother and fetus when used for isolation of fetal cells from pregnant women's blood samples and prenatal diagnosis of a broad spectrum of fetal cell chromosomal aberrations.     

血虚证动物模型的标准化研究初探

目的：建立标准化的血虚证动物模型。方法：采用慢性失血法、溶血法和骨髓抑制法制造三种血虚证模型,观察一般情况和与血发生有关的实验室指标,并用四物汤反证造摸效果。结果：三种动物模型均符合血虚证表现,同时出现外周血、骨髓有核细胞数目和细胞周期的改变,四物汤对3种动物模型均有一定疗效。结论：建立了3种符合标准化的血虚证模型。     

Effect of retinoic acid on the aggregation of erythrocytes and leukocytes in the blood

Human and animal blood smear staining with PAPh has revealed mononuclear leukocyte-erythrocyte aggregates. Administration of retinoic acid increased concentration and dimensions of these aggregates and was followed by preferential accumulation of PAPh-negative osmotically unstable erythrocytes. Similar changes were detected in the blood samples of women engaged in the production of retinoic acid. Aggregate concentration showed positive correlation with erythrocyte sedimentation rate and no correlation with prothrombin time.     

Immune-complex-trapping cells in the spleen of the oriental fire-bellied toad,Bombina orientalis

Summary The spleen of the oriental fire-bellied toad, Bombina orientalis, consists of well-developed white pulp, separated from the lymphocytic marginal zone by the connective tissue boundary layer. Injection of peroxidase-conjugated rabbit anti-peroxidase revealed that these immune complexes were localized on the surface of acid-phosphatase-positive and non-specific-esterasepositive cells in the white pulp. The majority of immunecomplex-trapping cells were present around the blood vessels. Cell processes of some of these cells penetrated into the wall of blood vessels. The significance of the present findings is discussed with respect to the evolution of immune-complex-trapping cells in the spleen.     

The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells

The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.     

Blood constituents during the estrous cycle and early pregnancy in dairy cows

The objective of this study was to determine if maternal platelet count, white blood cell count or other blood constituents undergo sustained alterations in concentration following fertilization. Blood samples from 17 Holstein females were collected over an 18-d period starting at estrus. Blood was analyzed for levels of platelets, white blood cells, red blood cells, hemoglobin and hematocrit. Results were analyzed for differences between nonpregnant and pregnant groups. Analysis of variance revealed a day-by-group interaction in the platelet count (P<0.01). White blood cell count showed both a day-by-group interaction and a difference between days (P<0.01). Red blood cell count, hematocrit and hemoglobin levels resulted in no significant difference between the two groups (P>0.05). While statistically significant differences were observed in platelet and white blood cell count, neither of these were sustained over a period longer than 2 d.     

Population kinetic study on the origin of guinea pig monocyte heterogeneity

Population kinetic studies were performed on guinea pig peripheral blood monocyte fractions isolated by counter-flow centrifugation elutriation following a single in vivo pulse of tritiated thymidine. Labeled large monocytes (volume 317 micron3; relative distribution 49%; circulating half-life 5.7 hr; and production rate 17,000 cells/ml blood/hr) accumulated in the circulation more rapidly, had a faster turnover time, and were produced in greater numbers than small monocytes (volume 283 micron3; relative distribution 34%; circulating half-life 10.8 hr; and production rate of 6100 cells/ml blood/hr). The kinetic data do not support a maturational sequence of small into large monocytes. Intermediate monocytes (volume 300 micron3; relative distribution 11%; circulating half-life 18.2 hr) and very large monocytes (volume 354 micron3; relative distribution 6%; circulating half-life 36.5 hr) had production rates, respectively, of only 1200 and 320 cells/ml blood/hr. Maxima in the labeling index curve for small and large monocytes suggested a generation time of 24 hr while grain count analysis revealed that these two cell fractions were derived from a precursor population with similar numbers of reductive divisions. Grain count analysis of intermediate and very large monocytes revealed that these cells differed from both small and large monocytes. Our data support the concept that monocyte subsets exist in guinea pig peripheral blood with different kinetics of production and survival.     

Chromosomal marker 20q-in cases of osteomyelosclerosis and CML

Summary Two male patients with myeloproliferative disorders (osteomyelosclerosis in one and CML in the other) were found to have a chromosomal marker 20q- in blood cells (unstimulated short time cultures). Marked erythropoietic disturbances were not revealed in these cases.The specificity of the 20q- marker for red cell disorders is discussed.     

Blood groups of macaques:a comparative study.

Distribution of the human-type and of the simian-type blood groups in rhesus, crab-eating, bonnet, pig-tailed and stump-tailed macaques revealed significant similarities and differences among these species. Human-type A--B-O blood groups cut across taxonomic lines and seem less value for taxonomic purposes than the simian-type blood groups detected by cross-reacting isoimmune rhesus monkey sera.     

Cytologic and immunologic prognosis of infertility. II Prognostic value of studies of immunoglobulins A,M, and G in peritoneal fluid and blood of infertile patients

Immunoglobulins of A, M, and G classes were studied in the blood and peritoneal fluid of 80 women during laparoscopic operation by nefelometry. An increased concentration of these immunoglobulins A, M, G was revealed in the patients with unfavourable prognosis. The immunological changes were seen to depend on the patient's age and the stage of lesion.     

Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Proton magnetic resonance study of the changes in blood lipids in malignant and benign neoplasms]

High-resolution 1H-NMR spectrometry (360 MHz) was used in combination with thin layer chromatography to study changes in composition and molecular structure of inverted micelles of blood lipids from patients with breast and uterus cancer. Changes in relative intensity of NMR signals of lipids compared with norm were revealed. The changes in the lipid composition of blood components, in particular, in cholesterol content, determine the differences in structure and molecular mobility of these lipids, which probably explains the regularities observed in the NMR spectra.     

Cell-mediated and humoral immunity in HBS-Ag carriers]

The presence of HBs--Ag in the blood was established in 1.8% of practically healthy persons, in 29.7% of virus hepatitis patients and in 86.0% of serum hepatitis patients. The immunological study of clinically healthy HBs--Ag carriers revealed a statistically significant decrease in the number of T and B lymphocytes and an increased level of serum IgA, IgM and IgG in the blood. Among donors, 7.6% were found to have lymphocytes sensitized to HBs--Ag and 15.4% were found to have lymphocytes sensitized to liver antigen, while for HBs--Ag carriers these data were 62.5% and 50%, respectively.     

High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia

Molecular methods were used to identify blood parasites frequently observed in blood smears of water pythons (Liasis fuscus) captured in our study area in the Northern Territory of Australia. A nested polymerase chain reaction (PCR) using primers amplifying the 18s ribosomal RNA (rRNA) nuclear gene resulted in a short PCR product (180 bp) matching this region in the genus Hepatozoon. However, because of the short sequence obtained. 2 new primers were designed based on 18s rRNA sequences of 3 Hepatozoon taxa available in GenBank. Using these primers, approximately 600 bp of the parasite's 18s rRNA gene was amplified successfully and sequenced from 2 water python samples. The new primers were used to investigate the prevalence of blood parasites in 100 pythons. In 25 of these samples we did not observe any blood parasites when examining stained slides. All the samples revealed a 600-bp PCR product, demonstrating that pythons in which we did not visually observe any parasites were infected by Hepatozoon spp. We also analyzed the nucleotide sequences of blood parasites in 4 other reptile taxa commonly encountered in our study area. The sequences obtained from water pythons and from 1 of these taxa were identical, suggesting that the parasite is capable of infecting hosts at different taxonomic levels.     

The distribution and origin of calcitonin gene-related peptide-containing nerve fibres in feline dental pulp

Summary The origin and distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in feline dental pulp were studied using indirect immunofluorescence. Nerve fibres with varicosities exhibiting CGRP-like immunoreactivity were observed to enter the pulp with blood vessels. Many CGRP-containing nerve fibres were found to extend along blood vessels in the central pulp, and some of these fibres exhibited a network arrangement in the walls of dental pulp blood vessels. However, some of fibres were apparently not associated with blood vessels. Some thin, CGRP-containing nerve fibres formed a part of the nerve plexus in the subodontoblastic area and penetrated into the odontoblastic layer. In animals that had undergone transection of the inferior alveolar nerve, no CGRP-containing nerve fibres were observed. Application of a double-immunofluorescence staining technique also revealed that the distribution of CGRP-containing nerve fibres is very similar to that of substance P-containing nerve fibres.     

Characterization of blood group active glycopeptides derived from porcine kidneys

Sialoglycopeptide fractions were prepared from the pronase digest of porcine kidneys by DEAE-Sephadex A-25 column chromatography and gel-filtration through Sephadex G-100. Their chemical compositions and large molecular size suggested that these glycopeptides were derived from mucin-type glycoprotein(s). The results of the beta-elimination reaction indicated that they have the O-glycosidic linkages between N-acetylgalactosamine and serine/threonine. The glycopeptides exhibited blood group A and H activities. The present study revealed that the porcine kidney contains the blood group antigens of glycoprotein nature.