Effects of Perfusion Rate on Permeability of Frog and Rat Mesenteric Microvessels to Sodium Fluorescein

The permeability, P _S, to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U , was varied over a range from 400 up to 2000–10 000 μm s⁻¹. P _S increased linearly with U . In 20 frog capillaries, mean (± S.E.M.) P _S (in μm s⁻¹) = 9.35 (± 1.55) U × 10⁻⁵+ 0.244 (± 0.0291). Similarly, in nine rat venules, mean P _S= 1.62 (± 0.385) U × 10⁻⁴+ 0.375 (± 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 μM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N ^G-nitro-L-arginine (20 μM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P _S, i.e. 85 % of the changes in P _S were changes in the permeability coefficient itself. A comparison between the changes in P _S with U and the previously described changes in microvascular permeability to K⁺ with U , suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.     

Increased Number of IgG Fc Receptors on Monocyte-Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (K_ass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of ¹²⁵I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 10⁴ FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 10⁴ FcR/cell). There was also a significant difference between the mean K _ass of the RA group and the control group-2.1±0.7 × 10³1/mol and 2.6±1.0 × 10³ 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 10⁴). Levels of circulating immune complexes (CIC) and of the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.     

Decreased peripheral blood γδ T cells in patients with bronchial asthma

Many cell populations are thought to be involved in the etiopathogenesis of bronchial asthma. We examined by flow cytometry the relative and absolute number of CD3*, CD4*, CD8*γδ TcR* T cells. CD19* B cells; and CD56* natural killer (NK) cells in the peripheral blood of 26 adult patients with difficult-to-control asthma (DCA) and 22 patients with minimally symptomatic asthma (MSA). Statistically higher relative and absolute numbers of NK cells (18.39±10.67% and 0.38±0.17×10⁹/l) in comparison with healthy controls (ll.77±8.06% and 0.25±0.19×10⁹/l) and significantly decreased relative and absolute numbers of γδ T cells (3.02±2.16% and 0.06±0.04×10⁹/l) in comparison with controls (5.65+2.90% and 0.13±0.08×10⁹/l) in the DCA patient group were found. After pooling of data from both MSA and DCA patients and dividing the patients according to the presence of allergy, the relative and absolute numbers of 78 T celts were found to be diminished in both the allergy (3.77±2.98 and O.O7±0.O5 ×10⁹/l) and nonallergy (3.06±1.78% and 0.06±0.03 ×10⁹/l) groups in comparison with healthy controls. The reason for the low number of 78 T cells in the peripheral blood of patients suffering from bronchial asthma is under investigation.     

GLUCOCORTICOID-INDUCED GROWTH INHIBITION OF HUMAN NEOPLASTIC SALIVARY GLAND DUCT CELL LINE (HSG)

The purpose of this study was to examine the effect of glucocorticoid on human neoplastic salivary duct epithelial cell line (HSG). Dexamethasone was found to inhibit cell growth and to increase cell size and the ratio of protein content to DNA content in a cell. The inhibition of cell growth was dose-dependent; in comparison to the control (33.8±3.1 h), the population doubling time was 1.57-fold longer in 10⁵ M dexamethasone (P<0.01, N-K test). [³H] thymidine incorporation was inhibited in 45.5% of the control at 10^-5 M. Plating efficiency was 20.5±3.0% in 10⁵ M and 47.0±4.4% in the absence of dexamethasone. Cell diameters increased 1.29 fold in 10^-5 M dexamethasone in comparison to the control size (16.0±2.1 μm). The ratio of total protein content of DNA content increased 1.46 fold in 10^-5 M dexamethasone-treated cells on the seventh day of cultivation. Scatchard plot analysis using [6, 7-³H] -triamcinolone revealed that the HSG cells had apparent cytosolic glucocorticoid receptors with an equilibrium dissociation constant (Kd value) of 6.48 nM, whose number of binding sites (NBS) was 57.8fmol/mg protein.     

Hypoxic preconditioning enhances renal superoxide dismutase levels in rats

Renal ischaemia releases reactive oxygen species (ROS) in the kidneys. We hypothesized that the kidneys are more resistant to the insult of ROS in chronically hypoxic rats. We thus compared rats kept at sea level (SL) and those that had been adapted to hypoxia (hypoxia adapted, HA) by exposure to an altitude of 5500 m in an altitude chamber for 15 h day⁻¹ for 4 weeks. Xanthine (X, 0.75 mg kg⁻¹) and xanthine oxidase (XO, 24.8 mU kg⁻¹) were injected intrarenally. A lucigenin-enhanced chemiluminescence method was employed to detect the amount of free radicals in renal venous blood samples and on the kidney surface. In the renal venous blood samples, 26.05 (± 4.36) × 10⁴ and 10.98 (± 1.79) × 10⁴ counts were detected in the SL and HA rats, respectively, after X-XO treatment; these figures were significantly different. On the kidney surface of the SL rats, the free radical count amounted to 12.77 (± 1.64) × 10⁴, while that in the HA rats was 8.47 (± 0.42) × 10⁴; these figures were also significantly different. There was a significant increase in urine volume and urinary excretion of Na⁺, K⁺ and protein after X-XO administration in both groups of rats. However, the effect was greater for the SL rats than for the HA rats. The lipid peroxidation of the kidneys was not significantly different in the two groups of rats. Finally, we found that the activity of superoxide dismutase (SOD) and SOD mRNA were higher in the renal tissue of HA rats. We conclude that the renal response to free radicals is attenuated after chronic hypoxia in rats, and that SOD might play an important role in protecting HA rats from oxidative stress.     

Kinetic Analysis of Subset Growth and B-Cell Activation in Pokeweed Mitogen-Stimulated Cultures of Peripheral Blood Mononuclear Cells Depleted of or Enriched for OKT8+ Cells

Peripheral blood mononuclcar cells (PBMN) that were depleted of OKT8⁺ cells and stimulated with pokeweed mitogen (PWM) produced higher cell yields and higher numbers of plaque-forming cells than unfractionated PBMN. Conversely, OKT8-enriched PBMN, prepared by mixing unfractionated and OKT8⁺ cells in a ratio of 3:1, gave reduced cell growth and B-cell activation. In OKT8-depleted cultures, B cells, OKT4⁺ cells, OKT8⁺ cells, and OKM1⁺ cells increased in number between days 4 and 7 of culture by factors of 9.8, 5.9, 20.1, and 5.6 respectively, whereas growth rates for these subsets were 2.4, 1.0, 2.0, and 1.3 in unfractionated cultures and 0.9, 1.0, 1.2, and 0.6 in cultures enriched for OKT8⁺ cells. On day 7 of culture, 73±10% of B cells secreted immunoglobulin in unfractionated cultures, whereas only 21±10% of B cells were activated in OKT8-enriched cultures. Surprisingly, PWM stimulation of OKT8-depleted PBMN produced only 40±12% activated B cells.     

Baroreflex-induced sympathetic activation does not alter cerebrovascular CO2 responsiveness in humans

We investigated the effect of baroreflex-induced sympathetic activation, produced by lower body negative pressure (LBNP) at −40 mmHg, on cerebrovascular responsiveness to hyper- and hypocapnia in healthy humans. Transcranial Doppler ultrasound was used to measure blood flow velocity (CFV) in the middle cerebral artery during variations in end-tidal carbon dioxide pressure ( P _ET,CO2) of +10, +5, 0, −5, and −10 mmHg relative to eupnoea. The slopes of the linear relationships between P _ET,CO2 and CFV were computed separately for hyper- and hypocapnia during the LBNP and no-LBNP conditions. LBNP decreased pulse pressure, but did not change mean arterial pressure. LBNP evoked an increase in ventilation that resulted in a 9 ± 2 mmHg decrease in P _ET,CO2, which was corrected by CO₂ supplementation of the inspired air. LBNP did not affect cerebrovascular CO₂ response slopes during steady-state hypercapnia (3.14 ± 0.24 vs. 2.96 ± 0.26 cm s⁻¹ mmHg⁻¹) or hypocapnia (1.31 ± 0.18 vs. 1.32 ± 0.19 cm s⁻¹ mmHg⁻¹), or the CFV responses to voluntary apnoea (+51 ± 19 vs. +50 ± 18 %). Thus, cerebrovascular CO₂ responsiveness was not altered by baroreflex-induced sympathetic activation. Our data challenge the concept that sympathetic activation restrains cerebrovascular responses to alterations in CO₂ pressure.     

II. The Effects of Thalidomide Treatment on Autoimmune-Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System

We describe here some immunomodulatory effects of thalidomide on autoimmune-prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5⁺μ^high, and in the numbers of total CD5⁺μ⁻, CD5⁻μ^high, CD5⁺μ^high lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5^lowμ^low pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5⁺ B cells and an oscillatory increase in the number of cells with CD5⁻ phenotype; (iv) a late reduction in the numbers of splenic total CD5⁺ B cells. These results are consistent with the notion that thalidomide controls a disease-associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.     

Complement (C3) Conversion by Rat Intestinal Glycoprotein and its Degradation Products

Several fragments obtained from alkaline borohydride degradation of a rat intestinal glycoprotein fraction have been tested for anti-complementary activity. Oligosaccharide alditols with a molecular weight of les than about 1 ± 10³ daltons showed no activity, whereas reduced oligosaccharides in the molecular weight range of about 1 ± 10³ to 3 ± 10³ daltons exerted a minor conversion of C3 by the alternative pathway. The low molecular weight fragments tested did not influence C3 conversion induced by the intact intestinal glycoprotein fraction. Of the fragments a peptide fraction, with an 'avenge' molecular weight of 2 ± 10³ daltons, and peptide-containing glycoconjugates exerted activation of C3 by both th classical and alternative pathways. Classical pathway activation by the intestinal glycoproteins depended on antibody, whereas alternative pathway activation did not. Alternative pathway activation appeared to require Factor B in that the intestinal glycoprotein induced no C3 conversion in serum heated to 50°C for 20 min. The rat intestinal glycoproteins had no protease activity on casein and no stimulating effect on human lymphocytes in vitro. Branching of oligosaccharide chains was not indicated by the methylation analyses carried out.     

A new estimate of the achondroplasia mutation rate

An estimate is derived of the mutation rate of achondroplasia, based upon the accumulated data of recent newborn studies in four cities. In a total of 242,257 births, seven infants had mutant achondroplasia, the diagnosis being confirmed radiologically in all but one. From this, the rate of mutation of the normal to the achondroplasia allele is calculated to be 1.4 × 10^--⁵± standard error 0.5 × 10^--⁵. Certain shortcomings of this estimate are discussed.     

Sustained Inhibition of Antigen-Induced Histamine Release from Human Lung by the β2-Adrenoceptor Agonist Terbutaline

Antigen-induced histamine release from passively sensitized human lung tissue was inhibited in the presence of the β₂-adrenoceptor agonist, terbutaline. A sustained and statistically significant suppression was detected in the concentration interval 3 × 10⁻⁸-1 × 10⁻⁶ M. Fifty per cent inhibition IC₅₀, was obtained at an interpolated concentration of 5.3 × 10⁻⁸± 0.4 × 10⁻⁸ M ( n = 13), when the histamine secretion was elicited with optimum concentration of antigen. Histamine release induced with a suboptimum concentration of antigen was inhibited to a greater extent than release initiated with optimum concentration. The data in the present investigation support the concept that terbutaline-induced inhibition of mediator release from human lung tissue can contribute to the clinical effectiveness of the drug during treatment of allergic asthma.     

Kinetics of the Reaction between a Monoclonal IgM Anti-I and Rabbit Red Cells

Rabbit red cells were found to bind a maximum of 1.8 × 10⁵ IgM anti-I-molecules. A K-value of 5.0 ± 0.8 × 10⁸ 1/M was determined for the reaction between the cells and intact anti-I at 5°C; the K-value was little affected by changes in temperature. Hybrid 17S and 8S molecules composed of varying amounts of cold agglutinin and inactive IgM were prepared by reduction with 2-mercaptoethanol and reassociation. The results obtained with the reassociated products indicate random reassociation at the half subunit level. It is concluded that a 17S fraction with four or more active half subunits shows binding similar to intact IgM, while 8S fractions with two active half subunits show binding similar to the cysteine-reduced 8S IgM.     

Effects of old age on human skeletal muscle energetics during fatiguing contractions with and without blood flow

We recently reported lower glycolytic flux (ATP_GLY) and increased reliance on oxidative ATP synthesis (ATP_OX) in contracting muscle of older compared to young humans. To further investigate this age-related difference in the pathways of ATP synthesis, we used magnetic resonance spectroscopy to determine the rates of ATP_OX, ATP_GLY and net phosphocreatine hydrolysis in vivo during maximal muscle contractions under free-flow (FF) and ischaemic (ISC) conditions in the ankle dorsiflexors of 20 young (27 ± 3 years; 10 male, 10 female) and 18 older (70 ± 5 years; 10 male, 8 female) adults. We hypothesized that ATP_GLY would be higher in young compared to old during FF contractions, but that old would be unable to increase ATP_GLY during ISC to match that of the young, which would suggest impaired glycolytic ATP synthesis with old age. Peak glycolytic flux during FF was lower in older (0.8 ± 0.1 m m ATP s⁻¹) compared to young (1.4 ± 0.1 m m ATP s⁻¹, P < 0.001) subjects. During ISC, peak ATP_GLY increased in old to a level similar to that of young (1.4 ± 0.2 m m ATP s⁻¹, 1.3 ± 0.2 m m ATP s⁻¹, respectively; P = 0.86), suggesting that glycolytic function remains intact in aged muscle in vivo . Notably, older adults fatigued less than young during both FF and ISC ( P ≤ 0.004). These results provide novel evidence of unimpaired in vivo glycolytic function in the skeletal muscle of older adults during maximal isometric dorsiflexion, and suggest a potential role for differences in metabolic economy and as a result, metabolite accumulation, in the fatigue resistance of the old.     

Receptor for α1-Microglobulin on T Lymphocytes: Inhibition of Antigen-Induced Interleukin-2 Production

The human plasma protein α₁-microglobulin (α₁m) was found to inhibit the antigen-induced interleukin-2 (IL-2) production of two different mouse T-helper cell hybridomas. α₁m isolated from human plasma and recombinant α₁m isolated from baculovirus-infected insect cell cultures had similar inhibitory effects. Flow cytometric analysis showed a binding of plasma and recombinant α₁m to the T-cell hybridomas as well as to a human T-cell line. Radiolabelled plasma and recombinant α₁m bound to the T-cell hybridomas in a saturable manner and the binding could be eliminated by trypsination of the cells. The affinity constants for the cell binding were calculated to be 0.4–1 × 10⁵  M ⁻¹ using Scatchard plotting, and the number of binding sites per cell was estimated to be 5 × 10⁵–1 × 10⁶. The cell-surface proteins of one of the T-cell hybridomas were radiolabelled, the cells lysed and α₁m-binding proteins isolated by affinity chromatography. SDS–PAGE and autoradiography analysis of the eluate revealed major bands with M _r-values around 70, 35 and 15 kDa. The results thus suggest that α₁m binds to a specific receptor on T cells and that the binding leads to inhibition of antigen-stimulated IL-2 production by T-helper cells.     

Variation in Gut-Homing CD27-Negative Lymphocytes in Inflammatory Colon Disease

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of α4β7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P  < 0.001), UC (12.2 ± 1.9%, P  < 0.001) and non-IBD colitis (10.5 ± 1.3%, P  < 0.01) as compared with controls (6.1 ± 0.5%). CD4⁺CD27⁻α4β7⁺ cells were increased in CD ( P  < 0.01). Lamina propria CD4⁺CD27⁻ populations were depressed significantly in CD ( P  < 0.05), UC ( P  < 0.02) and non-IBD colitis ( P  < 0.03). Mucosal CD4⁺CD27⁻ cells synthesized IL-4 in preference to interferon-γ. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.     

Peripheral T-cell Lymphomas Immunologic and Enzyme Histochemical Analysis of 15 Cases

Ffteen cases of peripheral T cell lymphoma were studied to evaluate the respective properties of various histologic types using enzyme histochemical and ultrastructural examinations in addition to immunological methods. Eleven cases in an ATLA negative group manifested various histologic patterns such as IBL like, pleomorphic and Lennert's lymphomas in comparison with the relatively monomorphic proliferation of neoplastic lymphoid cells in the 4 ATLA positive cases. The presence of neoplastic clear cells is characteristic of peripheral T-cell malignancies, and is likely to be found in CD4 lymphomas. There is an occasional reaction of epithelioid histiocytes and plasma cells with eosinophils, the former being designated Lennert's lymphoma and the latter IBL like T-cell lymphoma. Immunological examination revealed four immunophenotypic patterns: (1) CD2⁺3⁺4⁺8⁺, (2) CD2⁺ 3⁻4⁺8⁻, (3) CD2⁺3⁺4⁻8⁺, and (4) CD2⁺3⁺4⁺8⁺, but did not provide information concerning the intimate relationship between histologic types and immuno phenotyes. β-Glucuronidase reactivity, however, contributed to the distinction between helper and suppressor T cell malignancies, suggesting its usefulness for distinguishing these two cell types and their malignant counterparts.     

Rheumatoid Inflammatory T-Cell Clones express mostly Th1 but also Th2 and Mixed (Th0-Like) Cytokine Patterns

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1- or Th2-like functional subsets. Cytokine production was studied in 26 CD4⁺αβ⁺ and 2 CD8⁺αβ T-cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4⁺ clones were raised against various mycobacterial antigens and 11 CD4⁺ clones and 2 CD8⁺ clones were raised unspecifically using PHA and/or IL-2. The specificities of these clones are not known. In the mycobacterial antigen-specific group, all CD4⁺'αβ T-cell clones produced IFN-γ at high levels, while the production of IL-4 was generally absent or low (< 1 ng/ml), consistent with a Thl-like profile. Some of these clones, however, also produced various amounts of IL-10 which has been regarded as a Th2 product but can be produced also in lower amounts by Thi cells. One HSP-65-specific clone produced levels of IL-4 and IL-10 in the same order as that of IFN-γ, thus appearing to be Th0-like. Among the 11 unspecific CD4⁺ clones, 7 showed a Thl-like pattern but with lower levels of IFN-γ than the antigen-specific clones. However, three clones did not produce any IFN-γ activity but produced IL-4 and one of them also produced distinct amounts of IL-10, compatible with a Th2-like pattern. In addition, one of the clones also showed an almost equally strong IFN-γ and IL-4 production, thus most likely representing a Th0-like clone.