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.     

Evidence of porcine and human endothelium activation by cancer-associated carbohydrates expressed on glycoproteins and tumour cells

It is well established that after metastatic cancer cells escape the primary tumour and enter the circulation, their interactions with microvascular endothelium of a target organ constitute an essential rate-limiting step in haematogenous cancer metastasis. However, the physiological and biochemical processes supporting neoplastic cell arrest and retention in the microcirculation are still poorly understood. In this study, we present experimental evidence that microvascular endothelium of metastasis-prone tissues undergoes activation in response to desialylated cancer-associated carbohydrate structures such as Thomsen–Friedenreich (TF) antigen (Galβ1–3GalNAc) expressed on circulating glycoproteins and neoplastic cells. The metastasis-associated endothelium activation, manifested by marked increase in endothelial cell surface galectin-3 expression, causes gradual decrease in cancer cell velocities (from 72 × 10²± 33 × 10²μm s⁻¹ to 7.6 × 10²± 1.9 × 10²μm s⁻¹, mean ± s.d. ) accompanied by a corresponding increase in the percentage of rolling cells (from 3.3%± 1.2% to 24.3%± 3.6%, mean ± s.d. ), and results in human breast and prostate carcinoma cell arrest and retention in the microvasculature. This process, which could be of high importance in haematogenous cancer metastasis, was inhibited efficiently by an anti-TF antigen function-blocking antibody. Carbohydrate-mediated endothelial activation could be a process of physiological significance as it probably occurs in the interactions between a variety of circulating constituents and the vessel wall.     

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.     

Genetic polymorphism of esterase B3 in human leukocytes

Human tissues contain an esterase activity called ESB3, detectable by starch gel electrophoresis followed by staining with α-naphthyl butyrate. Using mononuclear leukocytes, we demonstrated an electrophoretic variant of ESB3. Family studies suggest that the variant is inherited as a simple Mendelian trait; individuals with the ESB3 2-1 phenotype are heterozygotes, designated ESB3 ¹ ESB3 ², to distinguish them from the more common homozygotes, ESB3 ¹ ESB3 ¹. The frequency of the ESB3 ² allele is estimated to be 0.035 in U.S. Whites. No homozygotes for this allele have yet been found.
Our studies suggest that the enzyme from ESB3 1 individuals exists primarily as a trimer of three identical subunits with a molecular weight of approximately 58000 daltons. The genetic variant ( ESB3 ² allele) appears to be the result of a mutation that does not affect the charge of the subunit, but rather reduces its ability to form and maintain the trimeric structure.     

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.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

Apolipoprotein A-IV is involved in detection of lipid in the rat intestine

Long chain triglyceride (>C12) in the intestinal lumen potently inhibits gastric emptying and acid secretion via the vagal afferent pathway. While the mechanism of inhibition involves the formation of chylomicrons, the essential role of the apolipoprotein apo A-IV is unclear. Using apo A-IV^−/− mice, we tested the hypothesis that inhibition of gastric emptying and gastric acid secretion in response to dietary lipid is dependent upon apo A-IV. As measured by nuclear scintigraphy in awake mice, gastric emptying of an ingested whole-egg meal was significantly faster in apo A-IV^−/− knockout versus A-IV^+/+ controls (34 ± 1 versus 54 ± 3 min, P < 0.0001). In anaesthetized A-IV^+/+ mice, meal-stimulated gastric acid secretion was 59% inhibited by intestinal lipid infusion; this was abolished in apo A-IV^−/− mice. Oral gavage of lipid in awake mice activated neurones throughout the nucleus of the solitary tract (NTS) in A-IV^+/+ mice, measured by immunohistochemical localization of Fos protein expression. However, in the mid region of the NTS (bregma −7.32 to −7.76 mm), Fos expression in response to intestinal lipid was significantly decreased by 50% in apo A-IV^−/− mice compared to A-IV^+/+ controls. We conclude that activation of the vagal afferent pathway and inhibition of gastric function in response to dietary lipid is partly dependent upon apo A-IV.     

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.     

Insulin and contraction increase nutritive blood flow in rat muscle in vivo determined by microdialysis of l-[14C]glucose

In the present study, a mathematical model using the microdialysis outflow: inflow (O/I) ratio of the novel analogue l -[¹⁴C]glucose has been developed which allows the calculation of the nutritive (and non-nutritive) flow in muscle as a proportion of total blood flow. Anaesthetized rats had microdialysis probes carrying l -[¹⁴C]glucose inserted through a calf muscle group (tibialis/plantaris/gastrocnemius). The nutritive fraction of total blood flow was determined under basal conditions and in response to contraction (electrical field stimulation), insulin (hyperinsulinaemic euglycaemic clamp with 10 mU min⁻¹ kg⁻¹ insulin) or saline control from limb blood flow and the microdialysis O/I ratio of l -[¹⁴C]glucose. Both contraction and insulin infusion decreased the O/I ratio of l -[¹⁴C]glucose and increased total limb blood flow. Calculations based on mathematical models using l -[¹⁴C]glucose O/I and limb blood flow revealed that during basal conditions, the nutritive fraction of total flow was 0.38 ± 0.06, indicating that basal flow was predominantly non-nutritive. Contraction and insulin increased the nutritive fraction to 0.82 ± 0.24 ( P < 0.05) and 0.52 ± 0.12 ( P < 0.05). Thus the increase in limb blood flow from insulin was fully accommodated by nutritive flow, while contraction increased nutritive flow at the expense of non-nutritive flow. This novel method using microdialysis and the O/I ratio of l -[¹⁴C]glucose allows the determination of the nutritive fraction of total flow in muscle as well as the proportion of total flow that may be redistributed in response to contraction and insulin.     

Determination of in vitro postantibiotic effects in Staphylococcus aureus and Escherichia coli by [3H]thymidine incorporation

Postantibiotic effects (PAE) and control-related effective regrowth time (CERT) of dicloxacillin, vancomycin, rifampin and gentamicin in Staphylococcus aureus and imipenem, gentamicin, tobramycin, doxycycline and rifampin in Escherichia coli were measured by standard viability counting and [³H]thymidine incorporation. For PAE determination, the two methods correlated well; r ² = 0.821 for S. aureus and r ² = 0.939 for E. coli . For viable counts below the detection limits of 10⁵ to 10⁶ log₁₀ CFU/mL, the PAE was overestimated by the [³H]thymidine method. Quantitation of CERT by both methods showed a good correlation, r ² = 0.867 for S. aureus and r ² = 0.997 for E. coli . Measuring [³H]thymidine incorporation in bacteria is a novel alternative method for the determination of PAE and CERT.     

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.     

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.     

Comparative Analysis of Peripheral Natural Killer Cells in the Two Phases of the Ovarian Cycle

Changes in endometrial Natural Killer (NK) cells during the luteal phase of the ovarian cycle are important in initiating/maintaining a subsequent pregnancy. In the present study it was investigated whether during the menstrual cycle changes occur also in peripheral blood (PB) NKs.

Method of study

Blood samples during the follicular and the luteal phase were collected from 30 women without fertility problems. Samples were analyzed by flow-cytometry for: (1) NK cells (CD3⁻CD16⁺CD56⁺) and (2) intracellular production of interferon-γ (IFN-γ) by NK cells. For the comparison and correlation of the two populations between the two phases, Wilcoxon signed-rank test and Spearman's Coefficient were used.

Results

The differences in percentages of CD3⁻CD16⁺CD56⁺ cells and that of CD3⁻CD16⁺CD56⁺/IFN-γ⁺ cells between the follicular and the luteal phase were not statistically significant (10.61 ± 5.11 versus 9.76 ± 4.57 and 6.48 ± 7.90 versus 7.30 ± 6.77, respectively, P  > 0.05). The correlation between the two variables (NK% and NK/IFN-γ%) was weakly positive ( P  = 0.07) only in the follicular phase.

Conclusion

The study did not reveal menstrual cycle-depended changes in PB NK cells. Thus, a suggestion to measure these cells in a specific phase of the cycle in order to predict the outcome of a subsequent pregnancy in women with fertility problems is objected.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

Protein kinase A is activated by the n–3 polyunsaturated fatty acid eicosapentaenoic acid in rat ventricular muscle

During cardiac ischaemia antiarrhythmic n –3 polyunsaturated fatty acids (PUFAs) are released following activation of phospholipase A2, if they are in the diet prior to ischaemia. Here we show a positive lusitropic effect of one such PUFA, eicosapentaenoic acid (EPA) in the antiarrhythmic concentration range in Langendorff hearts and isolated rat ventricular myocytes due to activation of protein kinase A (PKA). Several different approaches indicated activation of PKA by EPA (5–10 μmol l⁻¹): the time constant of decay of the systolic Ca²+ transient decreased to 65.3 ± 5.0% of control, Western blot analysis showed a fourfold increase in phospholamban phosphorylation, and PKA activity increased by 21.0 ± 7.3%. In addition myofilament Ca²+ sensitivity was reduced in EPA; this too may have resulted from PKA activation. We also found that EPA inhibited L-type Ca²+ current by 38.7 ± 3.9% but this increased to 63.3 ± 3.4% in 10 μmol l⁻¹ H89 (to inhibit PKA), providing further evidence of activation of PKA by EPA. PKA inhibition also prevented the lusitropic effect of EPA on the systolic Ca²+ transient and contraction. Our measurements show, however, PKA activation in EPA cannot be explained by increased cAMP levels and alternative mechanisms for PKA activation are discussed. The combined lusitropic effect and inhibition of contraction by EPA may, respectively, combat diastolic dysfunction in ischaemic cardiac muscle and promote cell survival by preserving ATP. This is a further level of protection for the heart in addition to the well-documented antiarrhythmic qualities of these fatty acids.     

The Allogeneic but not Syngeneic Dendritic Cells Effectively Generated Regulatory T Cells from Total Cd4+ Population without Exogenous Cytokines

Dendritic cells (DC) play a critical role in both the expansion of natural regulatory T cells (nTreg) and conversion of induced Treg (iTreg) from their precursors. In the present study, we evaluated the potential of DC to generate Treg from total CD4⁺ population which contains both nTreg and the precursors, and found that allogeneic (allo-DC) but not syngeneic DC (syn-DC) could effectively generated Foxp3⁺ Treg from total CD4⁺ population in the absence of exogenous cytokines. Compared with freshly purified CD4⁺ T cells, allo-DC-stimulated CD4⁺ T cells showed increased percentage of CD4⁺CD25⁺Foxp3⁺ Treg by 5–7-folds while syn-DC-stimulated CD4⁺ T cells did not. Furthermore, we demonstrated that the significant amounts of endogenous IL-2 and TGF-β, at least partially, contributed to the expansion of nTreg and conversion of iTreg in this cocultural system, respectively. Importantly, similar to nTreg, these allo-DC-generated Treg were capable of suppressing T cell response in vitro . Thus, our research provides a novel and efficient strategy for generation of Treg from total CD4⁺ population.     

Oligoclonality of TCRint Cells with a Low Diversity of TCR Complementarity-Determining Region 3 in Mice with Graft- Versus-Host Disease

Conventional T cells (i.e. TCR^high) are generated by the main stream of T-cell differentiation in the thymus. However, primordial T cells (i.e. TCR^int) are generated by extrathymic pathways and an alternative intrathymic pathway. Since TCR^int cells contain self-reactive clones, the diversity of the T-cell antigen receptor (TCR) complementarity-determining region (CDR) 3 was examined. The predominant Vβ8.2⁺ clones among TCR^int cells were selected for DNA sequencing. Thymectomized, irradiated mice subjected to bone-marrow transplantation (BMT) were used; graft-versus-host disease (GVHD), B6→(B6 × C3H/He) F ₁ and syngeneic BMT, B6→B6. In these combinations, only TCR^int cells were generated. Vβ8.2⁺ cells with a low diversity of CDR3 of V-gene expanded in GVHD mice. Vβ8.2⁺ cells of TCR^int and TCR^high cells in normal mice were polyclonal, showing that the former has a lower diversity of CDR3 than the latter. The clonality of activated TCR^high cells was examined, in which CD3^high cells (bm12 mice) were injected into 1 Gy-irradiated B6 nude mice. Some Vβ8.2⁺ clones among TCR^high cells were expanding but the diversity of CDR3 was greater than that of CD3^int cells, despite the fact that the recognition site of the H-2 difference was smaller. Taken together with invariant usage of Vα14, these results suggest that TCR^int cells have a low diversity of CDR3 of Vβ genes.     

Interstitial exclusion of albumin in rabbit lung during development of pulmonary oedema

The modifications of the macromolecular sieving properties of the pulmonary extracellular tissue matrix were studied in adult anaesthetized rabbits ( n = 10) exposed to increased tissue hydration. Exclusion of albumin from the perivascular pulmonary interstitial space was determined by using the continuous infusion method coupled with direct sampling of interstitial fluid performed through the wick technique. The rabbits underwent an intravenous infusion of saline amounting to 10 ( n = 5) or 20 % ( n = 5) body weight. Extracellular albumin distribution volume was derived from the steady state tissue concentration of radioactive rabbit serum albumin (¹²⁵I-RSA). Pulmonary extracellular and intravascular fluid volumes ( V _x and V _v, respectively) were measured as distribution volumes of ⁵¹Cr-EDTA and ¹³¹I-RSA, respectively, and interstitial fluid tracer concentrations were determined in interstitial fluid collected through implanted wicks. At the highest degree of hydration, interstitial fluid volume ( V _i= V _x− V _v) and extravascular albumin distribution volume ( V _a,w) significantly increased by 38.5 and 240.2 %, respectively, compared to control. Albumin-excluded volume ( V _e,a= V _i− V _a,w) was 398.9 ± 17 μl (g dry tissue weight)⁻¹; the albumin-excluded volume fraction ( F _e,a= V _e,a/ V _i) was 0.23 ± 0.01, 33.2 % of the control value. Data indicate that, at variance with what is observed in tissues like skin and muscle, pulmonary F _e,a is highly sensitive to tissue fluid content.