Observations on the development of descending pathways from the brain stem to the spinal cord in the clawed toad Xenopus laevis

Summary Anurans such as the clawed toad Xenopus laevis offer a unique opportunity to study the ontogeny of descending pathways to the spinal cord. Their transition from aquatic limbless tadpole to juvenile toad occurs over a protracted period of time during which the animal is accessible for experimental studies. In Xenopus laevis tadpoles the development of descending pathways has been studied from early limb-bud stage on (stage 50) with the aid of HRP slow-release gels. In stage 50, cells of origin of descending supraspinal pathways were already present throughout the reticular formation (including the interstitial nucleus of the fasciculus longitudinalis medialis) and in the vestibular nuclear complex. Also the giant Mauthner cells project to the cord at this stage. A spinal projection from the anuran homologue of the nucleus ruber of higher vertebrates does not appear before stage 58, i.e. when the hindlimbs are used for locomotion. Hypothalamospinal projections appear for the first time at stage 57. These observations in Xenopus laevis tadpoles suggest that reticulospinal and vestibulospinal projections innervate spinal segments very early in development, whereas the anuran red nucleus projects spinalward definitely later in development.     

A boussinesq model of natural convection in the human eye and the formation of Krukenberg's spindle

The cornea of the human eye is cooled by the surrounding air and by evaporation of the tear film. The temperature difference between the cornea and the iris (at core body temperature) causes circulation of the aqueous humor in the anterior chamber of the eye. Others have suggested that the circulation pattern governs the shape of the Krukenberg spindle, a distinctive vertical band of pigment on the posterior cornea surface in some pathologies. We modeled aqueous humor flow the human eye, treating the humor as a Boussinesq fluid and setting the corneal temperature based on infrared surface temperature measurements. The model predicts convection currents in the anterior chamber with velocities comparable to those resulting from forced flow through the gap between the iris and lens. When paths of pigment particles are calculated based on the predicted flow field, the particles circulate throughout the anterior chamber but tend to be near the vertical centerline of the eye for a greatest period of time. Further, the particles are usually in close proximity to the cornea only when they are near the vertical centerline. We conclude that the convective flow pattern of aqueous humor is consistent with a vertical pigment spindle. © 2002 Biomedical Engineering Society. PAC2002: 4266Ew, 8710+e, 8719Pp     

HSP70-Hom NcoI POLYMORPHISM AND HLA-ASSOCIATIONS IN THE FINNISH POPULATION AND IN PATIENTS WITH ANKYLOSING SPONDYLITIS OR REACTIVE ARTHRITIS

The NcoI polymorphism of the HSP70-Hom gene was investigated in the Finnish population and in two HLA-B27-associated autoimmune diseases. The two HSP70-Hom alleles were shown to be strongly associated to some specific HLA-B/DR haplotypes in random Finnish population and the segregation of the alleles as a part of these haplotypes was confirmed in 18 families. In addition, the HSP70-Hom alleles of 31 patients with ankylosing spondylitis (AS) and 38 with reactive arthritis (ReA) were compared with each other and with 56 unrelated healthy HLA-B27 positive individuals. The results indicated that the HSP70-Hom polymorphic variation is not connected independently to the different pathogenesis of AS and ReA, as no statistically significant differences between the patient groups and/or controls could be found. The HSP70-Hom status was investigated also in 28 homozygous HLA typing cells and when compared with previously published results of HSP70-1 and HSP0-2 polymorphisms, it appeared that these three MHC Class-III linked HSP70 genes segregated in fixed allelic combinations.     

Isolation of processed,H-2Kb -binding ovalbumin-derived peptides associated with the stress proteins HSP70 and GP96

Stress-induced proteins or heat shock proteins (HSP) of 96 kDa mass (gp96) and 70 kDa mass (HSP70) have been shown previously to elicit specific immunity to tumors from which they are isolated. This immunity is dependent on CD8⁺ cytotoxic T cells which are readily primed in vivo by immunization with HSP. The immunization capacity of HSP relies on their ability to bind antigenic peptides. Here we show that HSP70 and gp96 preparations purified from the ovalbumin (OVA)-transfected cell line E.G7 are associated with processed H-2K^b -binding peptides which contain the major H-2K^b -associated epitope SIINFEKL (OVA257 –264). Our data show for the first time in the well-defined OVA antigen system that not only endoplasmic reticulum-resident HSP, like gp96, are associated with processed antigenic peptides but that also the cytosolic HSP70 protein forms complexes with major finally processed MHC-binding epitopes.     

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As₂O₃) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As₂O₃ induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As₂O₃-treated infected cells as compared with As₂O₃-treated uninfected cells. Bcl-2 expression in As₂O₃-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As₂O₃-treated infected cells as compared with As₂O₃-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.     

Corticosterone stimulates the development of preoptic catecholamine neurons in tadpoles Bufo bufo japonicus

Summary In Bufo bufo japonicus catecholamine neurons in the preoptic recess organ (PRO) became detectable at the metamorphic climax by formaldehyde-induced fluorescence (Falck-Hillarp technique). In hypophysectomized tadpoles metamorphosis was inhibited and no fluorescent neurons appeared in the PRO. Implantation of a pituitary graft to the hypophysectomized tadpoles induced metamorphosis and development of PRO catecholamine neurons. Administration of corticosterone to hypophysectomized tadpoles resulted in the development of PRO catecholamine neurons in spite of the unmetamorphosed state. On the other hand, prolactin administration had no effect on the PRO neurons of hypophysectomized tadpoles. From these results, in conjunction with our previous results indicating that thyroxine treatment induces development of the PRO catecholamine neurons in thyroidectomized animals but not in hypophysectomized animals, it is concluded that corticosterone is a primary hormone for the development of PRO catecholamine neurons in toad tadpoles.     

Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites

Three monoclonal antibodies generated by immunization of mice withPlasmodium berghei-infected red blood cells were found to react with the 75-kDa heat-shock protein (HSP70) present in liver stages and crythrocytic forms of the parasites. These antibodies were shown to react with a recombinant protein encoding the carboxyl terminal half of PfHSP70 (aa 365–681). Differently from earlier results, we clearly demonstrated that HSP70 was also expressed in the sporozoite stage, using these monoclonal antibodies in an immunofluorescence and Western immunoblot assay. These monoclonal antibodies react not only with sporozoites ofP. berghei, the parasites originally used for the immunization, but also with sporozoites of several other rodent and human plasmodial species. Passive transfer of these monoclonal antibodies into naive mice, simultaneously injected with sporozoites, failed to neutralize the infectivity ofP. berghei sporozoites and to inhibit the development of liver stages ofP. yoelii.     

Intussusceptive microvascular growth in the lung of larval Xenopus laevis Daudin: a light microscope, transmission electron microscope and SEM study of microvascular corrosion casts

The remodeling of the uniform wide, plexus-like capillary bed of the lung of metamorphosing tadpoles of the South African clawed toad Xenopus laevis (Daudin) is studied from developmental stages 54 to 65 by scanning electron microscopy (SEM) of microvascular corrosion casts (VCCs), light microscopy (LM) and transmission electron microscopy (TEM). VCCs reveal that the remodeling of the existing uniform, plexus-like lung capillary bed into well-defined alveolar capillary meshworks starts in the caudal lung and then gradually proceeds cranially. Vascular remodeling is entirely by intussusceptive microvascular growth through insertion and enlargement of new and fusion of pre-existing capillary meshes. Analyses of lung tissue serial sections at the LM and TEM level confirm the presence of intracapillary cushions and tissue posts and correlate these structures in respect of size and location to the round to slit-like imprints and tiny ”holes” found in VCCs. Additionally, SEM of VCCs give clear evidence that intussusceptive microvascular growth is also involved in the remodeling and maturation of alveolar arterioles and venules. Accepted: 8 March 2000     

Immunohistochemical expression of C-myc oncogene,heat shock protein 70 and HLA-DR molecules in malignant cutaneous melanoma

The clinical course of malignant melanomas is frequently unpredictable, although a number of prognostically useful variables can be identified. There is a need for additional markers of prognostic value. In a series of 60 malignant cutaneous melanomas, we analysed the immunohistochemical expression of c-myc proto-oncogene, heat shock protein 70 (HSP70) and HLA-DR molecules in order to investigate their prognostic significance. C-myc, HSP70 and HLA-DR were expressed in 43.3%, 56.6% and 38.3% of all melanoma cases, respectively. Advanced Clark levels (Clark III–V) were significantly associated with c-myc expression rate (P<0.05), HSP70 detection (P<0.01) and HLA-DR positivity (P<0.01). Increased Breslow thickness (>1.5 mm) was related to HLA-DR expression (P<0.05). High mitotic rate was closely associated with c-myc positivity (P<0.05), while HSP70 and HLA-DR expression separately correlated to clinical stage of the disease (P<0.05). The evaluation of these variables may be of immunological and prognostic significance. They were found to be associated with melanocyte subpopulations of the vertical growth phase which are arguably characterized by an increased invasive potential.     

New method of <Emphasis Type="Italic">in vitro</Emphasis> culturing of pigment retinal epithelium in the structure of the posterior eye sector of adult rat

We propose a new method of organotypic roller 3D-culturing of the posterior sector of the eye. The method allows maintaining tissue viability in vitro for 14 days (which considerably surpasses the capacities of stationary culturing) and studying of the behavior of pigment retinal epithelial cells and choriocapillary membrane. Using this method we demonstrated phenotypic transformation, migration, and proliferation of pigment retinal epithelial cells under conditions of roller organotypic culture. In the absence of the retina, these cells exhibit properties of scavenger cells (phagocytes) both within and outside the layer. Under conditions of roller culturing in vitro, cells of the pigment retinal epithelium undergo changes similar to those observed in various retinal pathologies in vivo, including age-associated changes. Here we discuss the possibility of using the proposed method for evaluation of the effect of various factors added to the culture medium on the pigment epithelium, for modeling of processes developing in damaged pigment epithelium or under conditions of various pathologies, and for the study of regeneration responses in cells of pigment retinal epithelium in adult vertebrates. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 207–215, October, 2007     

蛙胚眼间质细胞对色素上皮分化前后的影响

本实验以泽蛙胚作材料。色素上皮分化前或分化后的眼泡经刮除间质细胞后,移植于同种蝌蚪体腔中培养,观察间质细胞对色素上皮分化前后的影响。结果证明,色素上皮分化前刮除间质细胞时,色素上皮不能发育或仅有片段色素上皮形成;而色素上皮开始分化或已经分化后刮除间质细胞时,色素上皮虽仍能发育和存在,但均脱离正常位置。这提示,间质细胞对眼色素上皮分化前后均有重要影响。而其作用性质似乎又有不同:在分化前,间质细胞的存在是色素上皮正常分化所必需的条件;在分化后,似乎起维持正常位置的机械作用。     

Cytoskeletal proteins bound to heat‐shock protein 70 may elicit resistance to simian immunodeficiency virus infection of CD4+ T cells

This study is based on the evidence that immunization of macaques with human CD4⁺ T cells elicits prevention of simian immunodeficiency virus (SIV) infection. We hypothesized that heat‐shock protein 70 (HSP70) isolated from CD4⁺ T cells may act as a chaperone and carry the protective host proteins. Two moieties of HSP70 were affinity‐purified from human CD4⁺ T cells; an ADP preparation with HSP70‐bound proteins (ADP‐HSP) and an ATP control preparation. Immunization of rhesus macaques with these preparations showed significant inhibition of SIVmac251 infectivity ex vivo in CD4⁺ T cells only with the ADP‐HSP (P = 0·01). Proteomic analysis identified three cytoskeletal elements, cofilin, profilin and γ‐actin, exclusively in the ADP‐HSP preparation. Investigation of the mechanism of prevention of SIV replication suggests that antibodies to the cytoskeletal proteins may inhibit actin depolymerization and facilitate viral degradation by the innate antiviral APOBEC3G. As cytoskeletal proteins are critical in the formation of virological and immunological synapses, finding specific antibodies and anti‐SIV/human immunodeficiency virus (HIV) factors suggests a novel insight into HIV‐1 immunopathogenesis.     

Stress for maintaining memory: HSP70 as a mobile messenger for innate and adaptive immunity

HSP are abundant and conserved proteins present in all cells. Upon temperature shock or other stress stimuli, HSP are synthesized intracellularly, which may protect cells from protein denaturation or from death. Although HSP are synthesized intracellularly, HSP can also be mobilized to the plasma membrane or even be released under stress conditions. Elucidating the roles of cell surface and extracellular HSP in immune regulation has attracted much attention in recent years. Extracellularly, HSP can serve a cytokine function to initiate both innate and adaptive immunity through activation of APC. HSP serves also a chaperone function and facilitates presentation of antigen peptide to T cells. Similarly, cell surface HSP may activate APC and promote antigen presentation through cell–cell contact. A study in this issue of the European Journal of Immunology demonstrates that cell surface HSP70 on DC induced by stress can upregulate membrane‐associated IL‐15, which in turn promotes the proliferation of CD4⁺CD45RA memory T cells. Moreover, a DC‐CD4⁺ T‐cell interacting circuit formed by CD40L on T cells and CD40 on DC is proposed to play a role in the maintenance of memory homeostasis. This study has widened our view of HSP in adaptive immunity as well as their classical functions such as APC activator and antigen carrier.     

Perspective on hepatic peroxisomes and pancreatic hepatocytes

Continued exposure to certain hypolipidemic drugs, plasticizers and herbicides leads to proliferation of peroxisomes in hepatic parenchymal cells. Sustained induction of peroxisome proliferation leads to the development of liver tumors in rats and mice. Peroxisome proliferator-induced pleiotropic responses, including the development of phenotypic properties in liver tumors induced by these agents, have been examined using autoradiography, immunofluorescence, immunoperoxidase, immunoelectron microscopy andin situ hybridization procedures in conjunction with northern blot and immunoblotting procedures. These studies confirmed that the biological effects of peroxisome proliferators were confined predominantly to liver cells and that the tumors appeared only in this organ. The cell specific effects of peroxisome proliferators have been documented in studies on the induction of peroxisome proliferationin vitro in primary liver cell cultures, in hepatocytes transplanted in subcutaneous fat or the anterior chamber of the eye in rats, and in rats with transdifferentiated pancreatic hepatocytes of the copper-deficiency model.     

Infection of retinal epithelial cells with L. amazonensis impacts in extracellular matrix proteins

One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis–RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis–ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis–ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite–host cell interactions.     

Changes in markers of muscle damage, inflammation and HSP70 after an Ironman triathlon race

We investigated the effects of an Ironman triathlon race on markers of muscle damage, inflammation and heat shock protein 70 (HSP70). Nine well-trained male triathletes (mean ± SD age 34 ± 5 years; V̇O_2peak 66.4 ml kg⁻¹ min⁻¹) participated in the 2004 Western Australia Ironman triathlon race (3.8 km swim, 180 km cycle, 42.2 km run). We assessed jump height, muscle strength and soreness, and collected venous blood samples 2 days before the race, within 30 min and 14–20 h after the race. Plasma samples were analysed for muscle proteins, acute phase proteins, cytokines, heat shock protein 70 (HSP70), and clinical biochemical variables related to dehydration, haemolysis, liver and renal functions. Muscular strength and jump height decreased significantly (P < 0.05) after the race, whereas muscle soreness and the plasma concentrations of muscle proteins increased. The cytokines interleukin (IL)-1 receptor antagonist, IL-6 and IL-10, and HSP70 increased markedly after the race, while IL-12p40 and granulocyte colony-stimulating factor (G-CSF) were also elevated. IL-4, IL-1β and tumour necrosis factor-α did not change significantly, despite elevated C-reactive protein and serum amyloid protein A on the day after the race. Plasma creatinine, uric acid and total bilirubin concentrations and γ-glutamyl transferase activity also changed after the race. In conclusion, despite evidence of muscle damage and an acute phase response after the race, the pro-inflammatory cytokine response was minimal and anti-inflammatory cytokines were induced. HSP70 is released into the circulation as a function of exercise duration.