Pharmacokinetics and physiological effects of intravenous hydromorphone in conscious dogs.

This study evaluated the pharmacokinetics, the sedative and anti-nociceptive effects of intravenous hydromorphone in dogs. Five adult dogs were administered hydromorphone (0.1 mg/kg and 0.2 mg/kg) and morphine (0.5 mg/kg and 1 mg/kg) at weekly intervals. Blood samples were drawn before and at 1, 2, 5, 15, 30, 60 and 120 min after drug administration. Plasma hydromorphone only was measured by high pressure liquid chromatography (HPLC) with electrochemical detection and pharmacokinetic parameters calculated. Anti-nociceptive and sedation scores were submitted to Kruskal-Wallis one-way anova on ranks and post-hoc Bonferroni test with 5% significance level. The data fitted a two-compartment model with a fast distribution (<1 min for both doses) and slower elimination rate. Mean elimination half-life was 80 +/- 52.7 and 57.7 +/- 30.4 min for the high and low dose, respectively. The apparent mean volumes of distribution at steady-state were 7.2 +/- 3 and 4.5 +/- 2.4 L/kg, while the clearance was 74.7 +/- 19 and 68.1 +/- 20 mL/kg/min for the high and low doses, respectively. Compared to saline, hydromorphone and morphine produced significant anti-nociception and sedation of similar magnitude for 120 min. In conclusion, intravenous hydromorphone has a large volume of distribution, and high clearance rate that exceeds hepatic blood flow. In dogs, it produced mechanical anti-nociception and sedation of a magnitude similar to morphine.

The specificity of the interaction between the agretope of an antigen and an Ia-molecule can depend on the T cell clonotype.

A series of T cell clones was developed from (B10 x B10.BR)F1 mice immunized with the isolated A chain of pig insulin. The T cell clones show considerable diversity as defined by their distinct reactivities to pig, beef, sheep and horse insulins in combination with the same syngeneic Ab alpha Ak beta molecules. These species variants of insulin differ from each other only in amino acid residues in position A8, A9 or A10 within the so-called A chain loop and responsiveness of mice to these variants is under Ir gene control. A detailed analysis of the stimulatory capacity of various insulin/Ia combinations including inhibition experiments with anti-Ia- and -L3T4 antibodies led to the following interpretation: the amino acid residues A8-A10 are involved in the interaction of the insulin A chain with the Ia molecules. This region can, therefore, be regarded as part of the agretope. Structural variations within this region can modify the stimulatory potency of the insulin variants. However, whether a particular amino acid substitution results in an enhancement or a reduction of the response depends on the fine specificity of the T cell clone involved. Thus, an interaction of Ia molecules with antigen cannot solely account for the functional specificity of an agretope, rather this also depends on the structure of the particular T cell receptor that participates in recognition.

Role of C3b receptors in the enhancement of interleukin-2-dependent T-cell proliferation.

The mechanism by which the complement system influences immune responses to T-cell-dependent antigens has not yet been clarified. That is why we studied the effect of the third complement component (C3) on different T-cell-dependent processes using well-defined mouse T-cell lines. While C3 did not influence the interleukin-2 (IL-2) production of the ST2/K-9 helper T-cells, the IL-2-dependent proliferation of the ST1 line was shown to be dose-dependently enhanced by C3. It is proved that neither the haemolytic activity of C3 nor the C3a fragment had any role in the process. The effect of C3 on the IL-2-dependent T-cell growth is even more enhanced (up to five-fold) when using polymerised C3. When the ST1 cell line is cultured in the presence of the cross-linked ligand, T-cells formed 80% less rosettes with red blood cells coated with antibody and mouse or human C3b. It is strongly suggested that C3--particularly when aggregated--exerts its enhancing effect on the growth of IL-2-dependent cell lines by binding to C3b receptors present on such T-cells.

Acidic "peptidophospholipids", a new class of hapten-bearing cell surface modifying reagents.

Covalent coupling of glutamyl-glutamic acid to the amino group of ether-phosphatidylethanolamine (EPE) yields an acidic "peptidophospholipid" (Glu2-EPE) which is water-soluble above pH 7.0 and stable to phospholipase A. The terminal amino group of Glu2-EPE is free for coupling with amino-reactive determinants. We describe the synthesis of various hapten-substituted peptidophospholipids as well as of an intermediate compound, coupled with the heterobifunctional reagent 3-(2'-pyridyl)-dithiopropionic acid N-hydroxysuccinimide ester. The latter derivative allows binding to sulfhydryl-containing molecules, e.g. peptides or proteins. So far, beef and pig insulin as well as trinitrophenyl-substituted ribonuclease A have thus been linked to Glu2-EPE. All derivatives of Glu2-EPE are water-soluble at physiological pH and readily adsorb to cell surfaces from aq. solution. Binding to cells is fast, stable and "non-toxic" over a wide range of concns. The adsorbed determinants are accessible to specific antibodies and facilitate complement-mediated cell lysis. Glu2-EPE thus appears to be a universal carrier molecule for fast, simple and mild modification of cells with foreign determinants, e.g. for Jerne plaque assays.

Presentation of insulin and insulin A chain peptides to mouse T cells: involvement of cysteine residues.

The requirements for insulin presentation and recognition by A alpha b A beta b- and A alpha b A beta k-restricted mouse T cells were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulatory for the T cells. This suggests that at least one of the Cys residues is essential for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for efficient presentation. Differences in stimulatory potency between the A chain peptides derived from the same insulin appear to be mainly due to the efficiency of uptake and/or processing by APC. Based on these findings we propose that processing in the case of insulin and its A chain derivatives involves the reductive deblocking of Cys residues or the rearrangement of disulfide bonds apart from a possible proteolytic cleavage. The same may apply to other proteins if Cys residues in the presented peptides are important for the interaction with Ia or the T cell receptor.

Perpetual proliferation of LYT-1 cells requires repetitive signals for IL-2 receptor induction by antigen-presenting cells.

T cell lines with specificity for bovine insulin and ovalbumin were maintained by serial stimulation with antigen presented on irradiated syngeneic spleen cells, alternating 3 days later with subculture in IL-2 containing medium (CM). When the cultures were repetitively split in CM, with concomitant dilution of antigen-presenting cells, a gradual loss of proliferative capacity of the cells in the presence of CM was observed. Absorption studies revealed a 20-fold reduction of IL-2 receptors on the surface of T blasts assayed 12 days after antigenic stimulation as compared with day 5 blasts. This decrement in the number of IL-2 acceptor sites reflected an actual decrease in cell surface density of IL-2 receptors. Restimulation of the T blasts with antigen and spleen cells induced both a substantial increase in IL-2 receptor density and responsiveness to CM. Furthermore, the permanent presence of antigen and spleen cells during splitting of the T blasts in CM prevented the loss of responsiveness to IL-2. As an interpretation we propose that the Lyt-1 cells studied here clear their IL-2 receptors from the cell surface after interaction with IL-2. Thus, each new round of replication of the daughter cells would be dependent on induction of IL-2 receptors by activating signals provided by antigen/Ia structures on accessory cells as well as possibly accessory cell products such as IL-1, rendering Lyt-1 cells sensitive to regulatory influences.

Characterization of a T cell-derived lymphokine that acts synergistically with IL 3 on the growth of murine mast cells and is identical with IL 4.

A mast cell-like cell line (SN-1) was established with the aid of growth factor(s) present in the supernatant of a Con A-stimulated L3T4+ T cell line. In analogy to other mast cell lines, IL 3 was identified as a growth factor for SN-1 cells. In addition, a second lymphokine produced by the T cells synergistically enhanced the IL 3-induced growth. This factor, originally termed mast cell growth enhancing factor (MaGEF), could be separated from IL 2, IL 3, and a CSF-like activity and was purified to homogeneity. The N-terminal amino acid sequence (8 residues) and the functional properties of this lymphokine proved to be identical with those reported for BSF-1 (IL 4). Unless applied at high concentrations, purified MaGEF did not stimulate growth of the SN-1 mast cells in the absence of IL 3. MaGEF was also found to act on two IL 2-dependent T cell lines by inducing significant thymidine incorporation which was suboptimal compared to that induced by IL 2 and which cannot be inhibited by anti-IL 2-antibodies. A panel of cell lines developed from mouse bone marrow with IL 3 or with a combination of IL 3 and MaGEF all reacted to MaGEF in the presence of IL 3 with considerably increased proliferation. It is therefore suggested that one of the physiological functions of MaGEF is to promote the recruitment of T-dependent mast cells.

Characterization of lymphokine-mediated activation of macrophages for antigen presentation: studies with long-term cultured bone marrow-derived macrophages and cloned T cells.

In cultures of bone marrow (BM) supplemented with L cell-derived colony-stimulating factor a pure population of macrophages (M phi) differentiates, which can be further propagated with a doubling time of 3.8 days. "Young" BMM phi obtained on day 8 of culture were shown to act as antigen-presenting cells inducing the antigen-specific proliferation of the cloned T cell line ST2/K.9, whereas "old" M phi had lost this ability. However, at any time tested (up to 132 days) the presentation function of old BMM phi could be completely restored by pulsing the cells with lymphokines (LK). A duration of 11 hr for the LK-pulse was sufficient to trigger the M phi to exert an optimal presentation function. This activity could be maintained when the LK-treatment was prolonged (tested up to 17 days). Activation was accompanied by a deceleration of growth. The LK effective in M phi activation were found to be contained in the supernatants of T cell lines stimulated by antigen or mitogen, and could be substituted by a low dose (5-10 units/ml) of recombinant interferon-gamma. In direct comparison LK-triggered BMM phi presented antigen as efficiently as peritoneal exudate M phi activated in vivo by ConA. Moreover, primed lymph node T cells responded to antigen-presenting BMM phi in a similar way as ST2/K.9 T cells. Therefore, these findings obtained with long-term cultured cells can be expected to reflect a physiological mechanism for the amplification of the immune response.

Co-development of naive CD4+ cells towards T helper type 1 or T helper type 2 cells induced by a combination of IL-12 and IL-4.

Cytokines were found to play a key role in Th cell differentiation. Among them IL-12 was shown to be a potent differentiation factor for Th1 cells, whereas IL-4 is the only known cytokine that promotes the development of Th2 cells. Upon addition of comparable amounts of IL-4 and IL-12 to a primary culture of naive CD4+ T cells activated by immobilized anti-CD3 mAb, it was found that the Th1-inducing capacity of IL-12 is dominated by the Th2-promoting effect of IL-4. However, high amounts of IL-12 (10,000 U/ml) in combination with low amounts of IL-4 (100 U/ml) led to the development of a Th cell population that, upon rechallenge, showed a substantial secondary IFN-gamma (Th1 cytokine) production concomitantly with the production of high amounts of IL-4 (Th2 cytokine). This can be due to the coexistence of Th1 and Th2 cells or to the development of Th0 cells producing a mixed pattern of cytokines. Immunofluorescence double staining of intracellular IL-4 and IFN-gamma in combination with flow cytometry (FACS) revealed that most of the emerging Th cells produced either IL-4 or IFN-gamma. Only a few double producers could be detected. This finding indicates that individual naive CD4+ T cells can differentiate under the same conditions towards Th1 or Th2 cells and implicates that the development of Th1 and Th2 cells is not necessarily mutually exclusive.

Characterization of the adjuvant effect of IL-12 and efficacy of IL-12 inhibitors in type II collagen-induced arthritis.

A destructive joint disease can be induced in susceptible DBA/1 mice by immunization with type II collagen emulsified with oil and either killed Mycobacterium tuberculosis or IL-12 as adjuvant. Cellular and humoral anti-collagen immune mechanisms appear to be involved in the pathogenesis of arthritis. We have characterized the adjuvant effect or IL-12 in more detail and addressed the question whether mycobacteria might act via the induction of endogenous IL-12. Injections of IL-12 into collagen-immunized DBA/1 mice promoted the development of IFN-gamma-producing CD4+ T cells and strongly upregulated the production of complement-fixing IgG2a and IgG2b antibodies resulting in severe arthritis. Neutralization of IFN-gamma in vivo largely inhibited the increase in antibody synthesis and prevented joint disease in IL-12-treated mice. However, collagen-specific IFN-gamma synthesis by T cells was further enhanced in these animals. Furthermore, IL-12 treatment promoted the development of IFN-gamma-producing T cells but failed to enhance antibody synthesis and to induce arthritis in C57BL/6 or BALB/c mice immunized with collagen in oil. These results indicate that the induction (by IL-12) of a strong collagen-specific T-cell response alone is not sufficient to trigger arthritis. Attempts to show a role for endogenous IL-12 in DBA/1 mice immunized with collagen with mycobacteria as adjuvant gave no reliable results. Whereas anti-IL-12 treatment delayed the onset and ameliorated the disease in some experiments, it failed to do so in other experiments, or, control reagents also had some effect. A slight inhibition of collagen-specific IgG2a synthesis was observed in most experiments in the sera of anti-IL-12-treated mice. Taken together, the results show that exogenous IL-12 can promote arthritis via its direct effect on T cells and its effect on antibody production, which is at least in part IFN-gamma-dependent. On the other hand, whether or not endogenous IL-12 is involved in the adjuvant effect of mycobacteria needs further clarification.

The monoclonal antibodies against mouse macrophages.

Five rat-mouse hybridomas producing monoclonal antibodies specific for mouse macrophages were obtained. The paper describes the hybridoma preparation and serological characterization of the monoclonal antibodies.

Further evidence for the genetic control of immune responsiveness to (T,G)-A--L by the B system in chickens.

The association of the higher immune response to (T,G)-A--L with B1 allele was confirmed in F2 generation birds of the cross between the high responding CB and low responding IC chicken inbred lines. No influence of the C and I blood group systems on the anti-(T,G)-A--L antibody production was observed.

Pharmacokinetics and physiological effects of two intravenous infusion rates of morphine in conscious dogs.

This study examined the pharmacokinetics and physiologic effects of two infusions rates of morphine in conscious dogs. Five adult dogs were randomly studied at weekly intervals. An initial dose of either 0.3 or 0.6 mg/kg were each followed by infusions of 0.17 and 0.34 mg/kg/h. Plasma morphine concentrations, physiological parameters, sedation and mechanical antinociception were evaluated during each infusion. Morphine was assayed by high pressure liquid chromatography (HPLC) with electrochemical coulometric detection and pharmacokinetic parameters were calculated. Data were fitted to a bi-compartment model with a rapid distribution (<1 min for both doses) and slower termination rate. For the high and low doses, respectively, mean+/-SD terminal half-life was 38+/-5 and 27+/-14 min, apparent volumes of distribution at steady-state were 1.9+/-0.5 and 1.3+/-0.8 L/kg, with clearances of 50+/-15 and 67+/-20 mL/kg/min. Steady-state plasma concentrations ranged from 93 to 180 ng/mL and 45 to 80 ng/mL in the high and low doses, respectively. Respiratory rate increased significantly, pulse oximetry remained>95% and body temperature decreased significantly during both infusions. No vomition or neuroexcitation occurred. Sedation and mechanical antinociception were both mild during the lower infusion rate, and mild to moderate during the higher infusion rate. In conclusion, morphine pharmacokinetics was not altered by increasing infusion rates, producing stable, long-lasting plasma concentrations.

Comparison of plasma histamine levels after intravenous administration of hydromorphone and morphine in dogs.

This study compared plasma histamine concentrations, behavioral and cardiovascular parameters following intravenous administration of hydromorphone and morphine in conscious dogs. Five adult female dogs received a 15-sec bolus injection of saline, hydromorphone (0.1 and 0.2 mg/kg) or morphine (0.5 and 1.0 mg/kg) randomly at weekly intervals. Blood samples were collected from the jugular vein before and at 1, 2, 5, 15, 30, 60 and 120 min after drug administration. Plasma histamine concentration, noninvasive oscillometric blood pressure, heart rate and rhythm were evaluated. Data were analyzed with repeated measures anova and Tukey-Kramer post hoc test with a 5% significance level. Median plasma histamine increased significantly only after the higher dose of morphine. Maximum plasma histamine measured was 0.8 ng/mL after saline and, after the lower and higher doses, respectively, 10.2 and 9.7 ng/mL for hydromorphone, and 440 and 589 ng/mL for morphine. One dog became hypotensive immediately after receiving the highest dose of morphine. Occasional ventricular premature contractions occurred in one dog after both opioids and dosages. No dogs vomited or defecated, but all salivated profusely with both opioids. Neuroexcitation occurred in four dogs following each opioid. In conclusion, intravenous hydromorphone induced minimal histamine release and was well tolerated by these conscious healthy dogs.

A blocked N-terminal residue in the light chain of rabbit immunoglobulin G.

The light chain of rabbit immunoglobulin G was shown to contain 15-20% blocked N-terminal residue. The blocked residue is pyrrolid-2-one-5-carboxylic acid, and most of the chains that contain this residue have the N-terminal sequence pyrrolid-2-one-5-carbonyl-valine.