Blood lymphocyte subsets in ATG-treated and allografted rats

Abstract. A single dose of rabbit antithymocyte globulin (ATG) was given as the sole immunosuppressive therapy in a model of strong MHC barrier rat heart allotransplantation. PVG/c hearts transplanted to Wistar/Kyoto (WKy) rats resulted in long-term surviving (LTS) grafts and cell-mediated lympholysis (CML) unresponsiveness in 50% of the animals. The effects of ATG treatment on the peripheral blood lymphocyte subsets were studied by flow cytometry. The absolute T-lymphocyte levels decreased to less than 5% and were normalized after 2 weeks. CD8-positive cells were normalized within 1 week, whereas CD4-and CD5-positive cells remained low. Rats with LTS grafts had low levels of all T-lymphocyte markers, especially the CD4-and CD5-positive cells. Rats rejecting their grafts showed an eightfold increase in levels of CD8-and CD5-positive lymphocytes and a twofold increase in levels of CD4-expressing lymphocytes. It is concluded that ATG treatment causes the immediate elimination of large lymphoid populations as well as long-lasting immunomodulation detectable in peripheral blood.     

Physiological pharmacokinetics of solutes in the isolated perfused rat hindlimb: Characterization of the physiology with changing perfusate flow,protein content,and temperature using statistical moment analysis

Distribution of Evans Blue (EB), sucrose, and water into the isolated perfused rat hindlimb was studied under various conditions using the multiple indicator dilution (MID) technique. Statistical moment analyses of the outflow profiles for the EB, sucrose, and water were used to define the vascular, extravascular, and total water spaces, respectively. The varied perfusion conditions included albumin content (2, 4.7, and 7%), temperature (25, 37, and 42 C), perfusate flow rate (2, 4, 8, and 12 ml/min) and the presence/absence of red blood cells. The range of studies undertaken were chosen to represent the variety of conditions used in the preparation of both isolated animal and human limbs, the latter being particularly important in cytotoxic therapy for recurrent malignant melanoma. The distribution volumes of EB, sucrose, and water were dependent on the flow rate and the albumin content of perfusate. The normalized variances (CV ² ) of the markers were of the following order: sucrose (2.18) > water (1.58) > EB (0.68), indicating that some disequilibrium occurs during the capillary exchange of water and sucrose. It is suggested that a Krebs-Henseleit buffer containing 2% BSA is a suitable perfusate for most studies of the isolated rat hindlimb perfusion. The effect of albumin concentration manifests itself only at higher flows.We acknowledge the support of the National Heart Foundation (Queensland) and the Mayne Bequest Foundation. This study was conducted while the investigator (Z.Y. Wu) was in receipt of a WHO Research Training Grant. Professor M. S. Roberts also acknowledges the support of the Queensland and Northern New South Wales Lions Kidney & Medical Research Foundation.     

Chromosomal Mapping of Genetic Locus Associated with Thymus-size Enlargement in BUF/Mna Rats

The thymoma-prone rat of the BUF/Mna strain is a useful model for human thymoma. In this strain thymoma development is regulated by a single autosomal susceptible gene, Tsr-1. At pre-thymoma age, BUF/Mna rats have extremely large thyrauses, when compared to those of other strains of rats. Genetic studies in crosses between BUF/Mna rats with large thymuses and WKY/NCrj rats with small thymuses suggested the presence of a major autosomal gene, Ten-1 , which contributes to thymus enlargement in a backcross population. Linkage studies between Ten-1 and microsatellite markers in backcross rats of (WKY/NCrj×BUF/Mna)Fl×BUF/Mna have led to the localization of Ten-1 in chromosome 1. This result may provide an approach to clone Tsr-1 , which could be allelic to Ten-1.     

Effects of chronic treatment with a nitric oxide donor on nerve conduction abnormalities and endoneurial blood flow in streptozotocin-diabetic rats

Abstract. This study examined the ability of nitrova-sodilator treatment with isosorbide dinitrate to prevent the development of reduced nerve conduction velocity and nutritive blood flow in streptozotocin-induced diabetes mellitus in rats. Two month untreated diabetes caused approximately 23% and 13% reductions in sciatic motor and saphenous nerve sensory conduction velocity ( P < 0.001). Isosorbide dinitrate treatment provided 64.6 and 67.6% protection for motor and sensory nerves, respectively ( P < 0.01). Sciatic endoneurial nutritive blood flow was measured by microelectrode polarography and a hydrogen clearance technique. After 1 month untreated diabetes, flow was reduced by 41.9% ( P < 0.001). Isosorbide dinitrate treatment for 1 month in non-diabetic and diabetic rats significantly increased blood flow ( P < 0.01). When between-group variations in blood pressure were taken into account, vascular conductance increased by 29% and 31% in non-diabetic and diabetic rats, respectively ( P < 0.01). Thus, nitrovasodilator treatment improves nerve perfusion and function in experimental diabetes, probably by compensating for reduced endothelium-derived nitric oxide release or action.     

The regulation of pulmonary vascular tone

1 The ability to manipulate pharmacologically pulmonary vascular tone independent of effects on systemic blood vessels is a desirable objective. Elucidation of the biochemical mechanisms underlying hypoxia-induced pulmonary vasoconstriction (HPV) may permit preferential targeting of the pulmonary circulation.
2 Here we review our studies of the role of locally synthesized candidate vasoactive factors in HPV. In addition, we present data demonstrating an attenuated pressor response to hypoxia in the pulmonary circulation of Fischer 344 rats compared with the Wistar-Kyoto (WKY) rat strain.
3 We propose that a systematic genome-wide search using the HPV phenotype and a panel of highly informative microsatellite markers will elucidate the genetic loci underlying the difference in susceptibility to HPV in these two rat strains and provide a valuable and novel insight into the factors that determine the HPV response.     

Quantitative analysis of spermatogenic DNA synthesis in the rat using a monoclonal anti-5-bromodeoxyuridine antibody

Summary. The utility of the 5-bromodeoxy-uridine (BrdUrd) labelling technique for the quantitative analysis of spermatogenic deoxyribonucleic acid (DNA) synthesis was investigated in the rat. Rat testicles were labelled by a single intraperitoneal injection of 100 mg kg⁻¹ of BrdUrd. The testicles were removed 1 h after injection, fixed in Bouin's fluid and embedded in paraffin. BrdUrd-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrdUrd antibody. The number of BrdUrd-labelled tubules per total number of tubules (percent L.T.), the number of BrdUrd-labelled cells per total number of tubules (tubular ratio) and the number of BrdUrd-labelled cells per number of Sertoli cells (Sertoli cell ratio in BrdUrd-labelled cells) were calculated as indices of spermatogenic DNA synthesis during each stage of the seminiferous epithelial wave. BrdUrd labelling was found exclusively in the nuclei of spermatogonia and in preleptotene spermatocytes in the seminiferous epithelium. The percent L.T. was generally greater than 50%, except in stages VI, VII and XIV, and the tubular as well as Sertoli cell ratios in BrdUrd-labelled cells was greater than 2.0 and 0.15, respectively, in stages I, II-III, V, VIII, X, and XII. The tubular ratio and Sertoli cell ratio in BrdUrd-labelled cells along the seminiferous epithelial wave had two distinct peaks. The distribution of the tubular ratio using the BrdUrd-labelling technique correlated well with the distribution previously established by measuring tritiated thymidine uptake per tubule. Thus, the BrdUrd labelling technique, which is more efficient than the tritiated thymidine labelling technique, can be used to quantitatively evaluate spermatogenic DNA synthesis.     

Age-Dependent Accumulation of Hybrid Vasopressin-Oxytocin Gene Products but not Hybrid Oxytocin-Vasopressin Products in the Endoplasmic Reticulum of Brattleboro Rats

The age-dependence of the incidence of magnocellular neurosecretory neurons containing abnormal accumulations of peptide in the rough endoplasmic reticulum was examined in homozygous Brattleboro rats and in their wild-type Long Evans counterparts. Neurons in which the immunophenotype of the peptide aggregates indicate that somatic cross-over mutations involving the 5' end of the vasopressin gene and the 3' end of the oxytocin gene have occurred, increased with age in homozygous Brattleboro rats, reaching a maximum of 24 cells per hypothalamus (approximately 0.6% of the vasopressin neurons). The increase occurred in both male and female animals but was significantly greater in females. The average incidence of such cells was 6 times greater in the supraoptic than in the paraventricular nucleus. No such cells could be detected in either nucleus of Long Evans rats despite the evidence for hybrid mRNA in these animals. Moreover, no accumulation of peptide translated from the hybrid mRNAs derived from the 5' end of the oxytocin gene and the 3' end of the vasopressin gene could be detected in either Brattleboro or Long Evans animals. These results strongly suggest that the accumulation of peptide in the rough endoplasmic reticulum of vasopressin neurons in homozygous Brattleboro rats is due to an abnormality other than the somatic crossing-over mutation. A second type of abnormal magnocellular neuron with accumulations of peptide in the rough endoplasmic reticulum, in which the immunophenotype of the peptide reveals products derived only from the oxytocin precursor, was present in both Long Evans and Brattleboro rats, but did not increase with age in Brattleboro rats. The incidence of these cells was similar in the supraoptic and paraventricular nuclei.     

Synergistic actions of the vitamin D analogue MC 1288 and 15-deoxyspergualin in xenotransplantation

Abstract: The vitamin D analogue MC 1288 (20-epi-1α,25-dihydroxycholecalciferol) effectively postpones rejection of cardiac, intestinal, skin, and aortic allografts. MC 1288 binds to the vitamin D receptor and is thus assumed to exert its immunosuppressive effects via the same mechanisms as 1α,25-dihydroxycholecalciferol, the active form of vitamin D. 1α,25-Dihydroxycholecalciferol has been demonstrated to inhibit the production of various cytokines (interleukin-2, interferon-γ, granulocyte macrophage colony-stimulating factor, and interleukin-12) and to prevent B lymphocyte secretion of immunoglobulins. In the present study MC 1288 was evaluated for its ability to prevent rejection of mouse-to-rat cardiac xenografts, alone and in combination with 15-deoxyspergualin (DSG). Combined treatment with MC 1288 (given days -1 to 9) and DSG (given day -1 and onward) postponed rejection from day 3.0 (untreated recipients) until day 19.5. In rats treated with MC 1288 or DSG as monotherapy, rejection occurred after 3.0 and 7.5 days, respectively. Functioning grafts, obtained on day 9 from recipients treated with MC 1288 and DSG in combination, displayed an almost normal morphology without any obvious deposition of immunoglobulins in the vessels of the grafts and with just a few infiltrating cells. Thus, we have demonstrated synergistic actions of MC 1288 and DSG in delaying rejection of xenografts. Analysis of cellular infiltration, immunoglobulin deposition and graft survival times in the various treatment groups indicate a combined inhibitory effect of these two drugs on the level of macrophage effector function, direct or indirect via T lymphocytes, as well as on antibody production.     

锌离子对大鼠海马突触体Ca^2＋—Mg^2＋ATP酶活性及蛋白合成的影响

行为实验己经证明，锌过多或缺锌均可影响脑功能。锌作为体内重要的微量元素，影响多种酶的活性及蛋白质和核酸的台成。本实验通过体外分离大鼠脑海马突触体，观察不同浓度锌离子对Ca2 －Mg2 ATP酶的活性和3H－Leu掺入突触蛋白合成的影响．结果表明：1．锌离子浓度在25μmol／L时增加该酶的活性（＜0．01），并促进3H－Leur掺入蛋白质的合成（＜0．05）。2．锌离子在50,100，200μmol／L的较高浓度时对Co2 －M2 ATP酶的活性有显著的抑制作用（分别为：P＜0.05．P＜0．01，P＜0.01），仅200μmol／L对3H—Leu掺入突触蛋白合成有抑制作用。本研究提示：适量的锌对突触体功能的维持是必要的，但剂量过高则起相反作用。     

Localization of Putative Receptors for Tetanus Toxin and Botulinum Neurotoxin Type A in Rat Central Nervous System

Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter release. These toxins act specifically on the nervous system by interacting with still non-identified protein receptors together with gangliosides. Whereas many biochemical data are available on their binding properties to neuronal membranes in vitro , there is poor morphological evidence of their binding to mammalian central nervous system. In the present study, the binding of tetanus and botulinum neurotoxin type A to rat brain sections is reported. Both toxins bound to nerve terminals with a broad distribution in brain. Tetanus toxin additionally bound to nerve fibres. The staining patterns were clearly shown to be due to the interaction of the heavy chains, which contain the binding moiety, with the tissue. In an attempt to investigate the nature of the acceptors present in the tissue, some sections were pre-incubated with periodic acid. This treatment resulted in the additional binding of botulinum neurotoxin type A to nerve fibres. Since the extended staining of nerve terminals was not modified by this pretreatment, it is suggested that protein receptors of clostridial neurotoxins are located at the nerve terminals, which may be common constituents of the synapses.