Studies of the blood of Ascidia ceratodes. Total blood cell counts, differential blood cell counts, hematocrit values, seasonal variations, and fluorescent characteristics of blood cells.

Seasonal, and animal size and weight variations of the blood cells of the vanadium-containing ascidian. Ascidia ceratodes, were determined. The fluorescent properties of various cell types were ascertained, and discussed in terms of cell development, phylogenic position of the species, and chemicals in the cells.     

Studies of the blood ofAscidia ceratodes. Total blood cell counts,differential blood cell counts,hematocrit values,seasonal variations,and fluorescent characteristics of blood cells

Summary Seasonal, and animal size and weight variations of the blood cells of the vanadium-containing ascidian,Ascidia ceratodes, were determined. The fluorescent properties of various cell types were ascertained, and discussed in terms of cell development, phylogenic position of the species, and chemicals in the cells.This investigation was supported by a grant from the Research Corporation and one of us (W.R.B.) received partial support from various University of California administrated fellowships. The current address of W.R.B. is Chevron Research Products, Richmond, CA. The use of the facilities at the Bodega Marine Laboratory, University of California are acknowledged.     

In vivo incorporation of 14C-phenylalanine into ascidian tunichrome.

Ascidia ceratodes exposed to 14C-phenylalanine in the surrounding seawater incorporates the radiolabel into newly biosynthesized tunichrome molecules. Radioactivity can be detected in tunichrome extracted from circulating blood cells within one day following initial exposure to the radiolabel; weak activity (less than or equal to 4 microCi/mol tunichrome = 22 nmol phenylalanine/mol tunichrome) is detected in 1 to 10 days; significantly higher amounts of radiolabel (57 microCi/mol tunichrome = 318 nmol phenylalanine/mol tunichrome) appear 20 days after seawater exposure. Therefore, phenylalanine can function as a precursor in the biosynthesis of tunichrome.     

In vivo incorporation of14C-phenylalanine into ascidian tunichrome

Ascidia ceratodes exposed to¹⁴C-phenylalanine in the surrounding seawater incorporates the radiolabel into newly biosynthesized tunichrome molecules. Radioactivity can be detected in tunichrome extracted from circulating blood cells within one day following initial exposure to the radiolabel; weak activity (4 Ci/mol tunichrome=22 nmol phenylalanine/mol tunichrome) is detected in 1 to 10 days; significantly higher amounts of radiolabel (57 Ci/mol tunichrome=318 nmol phenylalanine/mol tunichrome) appear 20 days after seawater exposure. Therefore, phenylalanine can function as a precursor in the biosynthesis of tunichrome.     

Ultrastructural study of vanadocytes in Ascidia malaca

Summary The ultrastructural aspects of vanadocytes, found in the circulating blood of Ascidia malaca, were studied in formaline-fixed material. The results indicate that the diverse morphological aspects of vanadophores reported here, are probably concerned with different metabolic stages of vanadine.This work was supported by a grant from the University of Naples at the Zoological Station of Naples.     

Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.     

The effect of shock on blood oxidation-reduction potential.

Oxidation-reduction (redox) potential measurements were made in the blood of rabbits subjected to hemorrhagic shock followed by treatment with a mild oxidizing agent (albumin). Control redox potential reading corrected for pH was -8.8 +/- 1.3 millivolts (mV) in arterial blood (A) and -18.0 +/- 2.0 mV in venous blood (V). This A-V difference indicated that hydrogen equivalents coming from muscle and other tissues were partially consumed in the lungs. A 20-mV drop on the V and a 13 mV on the A side was seen after shock. This did not fully return to control 2 h after return of the shed blood. Infusion of 2 g of albumin/kg/h raised the V redox potential to control, but it returned to untreated levels when the albumin was discontinued. The reductive load imposed on the animal by shock appeared to be large and not readily reversed by reperfusion or by the quantity of albumin given. Thus, it may be concluded that cellular respiration had not been adequately restored. This reductive load may impede recovery by suppression of cellular respiration and other cell and organ functions.     

The effect of shock on blood oxidation-reduction potential

Oxidation-reduction (redox) potential measurements were made in the blood of rabbits subjected to hemorrhagic shock followed by treatment with a mild oxidizing agent (albumin). Control redox potential reading corrected for pH was –8.8±1.3 millivolts (mV) in arterial blood (A) and –18.0±2.0 mV in venous blood (V). This A-V difference indicated that hydrogen equivalents coming from muscle and other tissues were partially consumed in the lungs. A 20-mV drop on the V and a 13 mV on the A side was seen after shock. This did not fully return to control 2 h after return of the shed blood. Infusion of 2 g of albumin/kg/h raised the V redox potential to control, but it returned to untreated levels when the albumin was discontinued. The reductive load imposed on the animal by shock appeared to be large and not readily reversed by reperfusion or by the quantity of albumin given. Thus, it may be concluded that cellular respiration had not been adequately restored. This reductive load may impede recovery by suppression of cellular respiration and other cell and organ functions.     

Inhibition of endogenous phosphodiesterase 7 promotes oligodendrocyte precursor differentiation and survival

During the development of the central nervous system (CNS), oligodendrocyte precursors (OPCs) are generated in specific sites within the neural tube and then migrate to colonize the entire CNS, where they differentiate into myelin-forming oligodendrocytes. Demyelinating diseases such as multiple sclerosis (MS) are characterized by the death of these cells. The CNS reacts to demyelination and by promoting spontaneous remyelination, an effect mediated by endogenous OPCs, cells that represent approximately 5–7 % of the cells in the adult brain. Numerous factors influence oligodendrogliogenesis and oligodendrocyte differentiation, including morphogens, growth factors, chemotropic molecules, extracellular matrix proteins, and intracellular cAMP levels. Here, we show that during development and in early adulthood, OPCs in the murine cerebral cortex contain phosphodiesterase-7 (PDE7) that metabolizes cAMP. We investigated the effects of different PDE7 inhibitors (the well-known BRL-50481 and two new ones, TC3.6 and VP1.15) on OPC proliferation, survival, and differentiation. While none of the PDE7 inhibitors analyzed altered OPC proliferation, TC3.6 and VP1.15 enhanced OPC survival and differentiation, processes in which ERK intracellular signaling played a key role. PDE7 expression was also observed in OPCs isolated from adult human brains and the differentiation of these OPCs into more mature oligodendroglial phenotypes was accelerated by treatment with both new PDE7 inhibitors. These findings reveal new roles for PDE7 in regulating OPC survival and differentiation during brain development and in adulthood, and they may further our understanding of myelination and facilitate the development of therapeutic remyelination strategies for the treatment of MS.     

Ultrastructural localization of vanadium in the blood cells of Ascidiacea

Summary X-ray histospectrographic analysis at the scanning and transmission electron microscope (STEM) are made on the blood cells ofPhallusia mamillata Cuvier andCiona intestinalis, to study the direct intracellular sites of accumulation of vanadium. The results show a clear accumulation of vanadium on the membrane and in the granules of vacuoles of amebocytes, signet ring cell, compartment cell and traces of metal in the vanadophores of vanadocytes.We thank Dr C. Weichan and Mr G. Hubert for kind hospitality and for technical assistance in the Department of Electron Microscopy of Siemens Laboratory in Karlsruhe (BRD). We thank also Dr V. Andria of Siemens Elettra in Rome (Italy).     

Loss of antibody production accompanied by chromosome loss in a cloned hybrid line secreting antibodies to sheep red blood cells

Somatic cell hybrids between Sp2/O-Ag14 mouse myeloma cells and lymphocytes derived from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were produced. One hybrid producing IgG1 antibody to SRBC was selected, cloned twice and subsequently transferred to BALB/c mice. After a number of transfers it was found that the antibody titer in ascites fluid gradually decreased. Cytogenetic analysis revealed gradual chromosome loss in the hybrid clone, which produced progressively less antibody.     

GSK-3β is essential for physiological electric field-directed Golgi polarization and optimal electrotaxis

Endogenous electrical fields (EFs) at corneal and skin wounds send a powerful signal that directs cell migration during wound healing. This signal therefore may serve as a fundamental regulator directing cell polarization and migration. Very little is known of the intracellular and molecular mechanisms that mediate EF-induced cell polarization and migration. Here, we report that Chinese hamster ovary (CHO) cells show robust directional polarization and migration in a physiological EF (0.3–1 V/cm) in both dissociated cell culture and monolayer culture. An EF of 0.6 V/cm completely abolished cell migration into wounds in monolayer culture. An EF of higher strength (≥1 V/cm) is an overriding guidance cue for cell migration. Application of EF induced quick phosphorylation of glycogen synthase kinase 3β (GSK-3β) which reached a peak as early as 3 min in an EF. Inhibition of protein kinase C (PKC) significantly reduced EF-induced directedness of cell migration initially (in 1–2 h). Inhibition of GSK-3β completely abolished EF-induced GA polarization and significantly inhibited the directional cell migration, but at a later time (2–3 h in an EF). Those results suggest that GSK-3β is essential for physiological EF-induced Golgi apparatus (GA) polarization and optimal electrotactic cell migration.     

Free amino acid composition of the intestinal contents,intestinal cells and hemolymph ofPhilosamia cynthia larvae

Summary Free amino acid composition of the intestinal contents, intestinal cells and hemolymph has been determined in larvae of the mothPhilosamia cynthia. From the hemolymph/lumen concentration ratio, an active transport could be inferred for neutral and basic amino acids. The values of cell/lumen and hemolymph/cell ratios suggested that the active step in the transport mechanism could be localized at the luminal pole of the enterocyte for neutral amino acids (except aromatic amino acids) and at the basolateral pole of the enterocyte for basic amino acids (except arginine).This work was supported by grants from Italian Consiglio Nazionale delle Ricerche and from Ministero della Pubblica Istruzione, Rome. The authors are indebted to Prof. V. Capraro for helpful discussion.     

Loss of antibody production accompanied by chromosome loss in a cloned hybrid line secreting antibodies to sheep red blood cells

Summary Somatic cell hybrids between Sp2/O-Ag14 mouse myeloma cells and lymphocytes derived from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were produced. One hybrid producing IgG₁ antibody to SRBC was selected, cloned twice and subsequently transferred to BALB/c mice. After a number of transfers it was found that the antibody titer in ascitec fluid gradually decreased. Cytogenetic analysis revealed gradual chromosome loss in the hybrid clone, which produced progressively less antibody.     

Bacterial lysis of cytoplasm of Cosmarium,a green alga

Summary The protoplast of the green alga Cosmarium is lysed by a coccoid bacterium, whereas the cell wall remains intact. Pyrenoids are more resistant to lytic action than the rest of the chloroplast. Different stages of the lysis are described.We thank the Heads of the Botany and Soils Departments for providing laboratory facilities. Thanks are also due to Dr V. R. P. Garg for help in photography.     

Induction of dolykaryocytes by vesicular stomatitis virus in XC rat cells]

V.S.V. induced polycaryocytes in rat embryonic fibroblasts, transformed by the Prague strain of Sarcoma Rous (XC cells). This fusion is strictly dependent on the expression of the viral genome and is probably due to the incorporation of viral antigens in the cell membrane. The integrity of cellular RNA synthesis is however not required. The fusion is probably due to a membrane structure characteristic of these transformed cells.     

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.     

Fluorescein-concanavalin A conjugates distinguish between normal and malignant human cells: a preliminary report

A method is described for using a fluorescein isothiocyanate concanavalin A conjugate to stain human cell membranes in formalin fixed paraffin embedded tissue. 57 neoplastic and normal tissue sites were examined. In 54 malignant tumours, bright green fluorescence was confined to the cell membranes while in 23 benign tumours and normal tissue sites, the membranes were unstained or showed a diminished level of fluorescence. The distinction between malignant and hyperplastic or normal cells was clear cut and definite.     

Detection of human cytomegalovirus (HCMV) DNA from cell-free plasma of immunosuppressed patients by nested PCR: influence of nucleic acid extraction method

Conclusions Blood cells and plasma preparations from HCMV-seropositive healthy blood donors were all nPCR negative. Detection of HCMV DNA from PBMC and granulocytes (DNAemia) of immunosuppressed patients by nPCR did not correlate with the isolation of infectious virus from these cell populations in cell culture (viremia). However HCMV could be isolated in 60% of cases from other materials of the same patient. HCMV DNA detected in blood cells persisted for up to one year in an asymptomatically infected individual after NTX. The sensitivity of HCMV DNA detection in cell-free plasma (up to 5 fg) depended on the method used for DNA isolation. The rate of HCMV DNA detection in plasma was lower than in leukocytes. In all cases of positive plasma PCR infectious virus could be isolated from any other material of the symptomatically infected patients. Therefore HCMV DNA PCR from plasma of immunosuppressed patients seems to be a suitable and easy alternative to HCMV RT/PCR for routine diagnosis of HCMV disease.