Immune enhancing effect of a growth hormone secretagogue.

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.

Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels.

The voltage-gated potassium channel, Kv1.3, is specifically expressed on human lymphocytes, where it controls membrane potential and calcium influx. Blockade of Kv1.3 channels by margatoxin was previously shown to prevent T cell activation and attenuate immune responses in vivo. In the present study, a triterpene natural product, correolide, was found to block Kv1.3 channels in human and miniswine T cells by electrophysiological characterization. T cell activation events, such as anti-CD3-induced calcium elevation, IL-2 production, and proliferation were inhibited by correolide in a dose-dependent manner. More potent analogs were evaluated for pharmacokinetic profiles and subsequently tested in a delayed-type hypersensitivity (DTH) response to tuberculin in the miniswine. Two compounds were dosed orally, iv, or im, and both compounds suppressed DTH responses, demonstrating that small molecule blockers of Kv1.3 channels can act as immunosuppressive agents in vivo. These studies establish correolide and its derivatives as novel immunosuppressants.

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.

Surgical process scheduling: a structured review.

There is no generally accepted definition of surgical process scheduling available in the literature; nursing researchers, physicians, administrators, and management scientists each view scheduling differently. To overcome this communication problem, a number of authors have proposed conceptual frameworks for surgical process scheduling. These frameworks have unfortunately been either unsatisfactory or incomplete. In this paper, we describe a conceptual framework for surgical process scheduling and use it to classify the existing literature. Results from the review indicate that while operational aspects of advance and allocation scheduling are well understood, further research should be directed towards resolving scheduling issues at strategic and administrative levels. In addition, techniques for integrating operating room (OR) scheduling with other hospital operations are required.

Association of serine protease with the rise of intracellular calcium in cytotoxic T lymphocytes.

The precise role of the granular enzyme A (granzyme A), a serine protease, in the lytic process of cytotoxic T lymphocytes (CTL) is not clear. We have recently constructed a CTL line transfected with the antisense gene of granzyme A (a-GrA). These a-GrA CTL had lower GrA activity as well as decreased lytic activities, as measured by 51Cr and by DNA degradation assays. Furthermore, at low effector:target ratio (1:8) in prolonged lytic assays, they could not lyse targets as rapidly as the control CTL. When we examined their ability to exocytose BLT (CBZ-L-lys-thiobenzyl)-esterase in the presence of anti-CD3 antibody, the a-GrA CTL exocytosed poorly compared to the parental CTL or control transfectant with a CAT gene. Most strikingly, a-GrA cells could not release intracellular stores of Ca2+ in response to anti-CD3 induction, although the Ca2+ flux was normal when they were stimulated with ionomycin. When the parental CTL was treated with a specific benzyllactam inhibitor of BLT-esterase or N-tosyl-L-phenylalanylchloromethyl ketone, the Ca2+ flux induced by anti-CD3 was also suppressed. We propose that granzyme A is involved in the signal transduction pathway that causes the rise of the intracellular calcium.

Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.

The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).

Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity.

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.

The enzymatic activity of human cytotoxic T-lymphocyte granzyme A and cytolysis mediated by cytotoxic T-lymphocytes are potently inhibited by a synthetic antiprotease, FUT-175.

The synthetic antiprotease, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), was found to be an extraordinarily potent and rapid inhibitor of human Q31 cytotoxic T-lymphocyte granzyme A. The granzyme A was inhibited in a time-dependent manner with kobs/i = 430,000 +/- 80,000 M-1 s-1. Four other FUT-175 analogs were also found to be potent, rapid Q31 granzyme A inhibitors. All five compounds inhibited Q31 cytotoxic T-lymphocyte-mediated cytolysis of human JY lymphoma cells, but at concentrations far in excess of those needed for granzyme A inhibition. The data presented suggest that postmarketing surveillance of FUT-175 should include a review of possible immunosuppressive side-effects, such as increased susceptibility to viral infections and to neoplastic transformations.

Clinical signs and bone changes associated with phosphorus deficiency in beef cattle.

For 10 years, 42 female Herefords (as they progressed from weanling calves to aged cows) were fed diets individually, with phosphorus (P) content being the only variable. During growth and the first 3 gestations, clinically evident differences were not associated with 2 dietary treatments (approx 12 and 38 g of P/day). During the next 2 gestations (2 years), half the cows from each original treatment group were fed less than 6 g of P (n = 21 cows, 11 from the group fed 12 g of P/day and 10 from the group fed 38 g of P/day) daily. The other half were fed diets supplying approximately 8 g of P (n = 11 cows fed 12 g of P/day) and 35 g of P (n = 10 cows fed 38 g of P/day) daily. During the last 3 years of the experiment, all remaining cows were fed diets containing 12 g (n = 19 cows originally fed 12 g) or 19 g (n = 17 cows originally fed 38 g) of P/day. Cows fed diets containing less than 6 g of P/day developed an insidious and subtle complex syndrome characterized by weight loss, rough hair coat, abnormal stance, and lameness. Spontaneous fractures occurred in the vertebrae, pelvis, and ribs. In severely affected cows, fractures did not heal properly. Some bones were demineralized markedly, and the cortical surfaces were porous, chalky white, soft, and fragile. Osteoid tissue was not properly mineralized. Radiography revealed diminished bone density (osteoporosis), cortical thinning, and resorption of trabeculae. Time-related availability of dietary P initiated excessive turnover of bone, with resultant structural changes and impaired function.(ABSTRACT TRUNCATED AT 250 WORDS)

Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity.

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.

Dietary phosphorus for beef cows.

Hereford heifers (48 initially) were individually fed variable amounts of dietary phosphorus (P) from weaning through their 8th gestation. During phase I, 2 groups (24 cows each) were given 20.6 to 38.1 g of P/day and 6.0 to 12.1 g of P/day (increasing as animals grew). During phase II (the 4th gestation), half of the animals from each group were maintained with the same respective diets, and the other half were given 5.1 to 6.6 g of P/day. Within 6 months, animals given 5.1 to 6.6 g of P/day gradually developed signs of P deficiency. Clinical signs of deficiency and lesions included general unthriftiness, body weight loss, reduced feed consumption, reluctance to move, abnormal stance, spontaneous bone fractures, and finally, impaired reproductive performance. Cows given 7.8 to 8.9 g of P/day in diet 2 during phase II did not develop discernible clinical signs of P deficiency. Cows that were given 5.1 to 6.6 g of P/day apparently regained their health and reproductive capability when fed 11.7 to 12.6 or 17.1 to 20.5 g of P/day. The data indicate that bones serve as an effective storage source of P and support and buffer body needs until there is serious depletion of skeletal P. These results indicate that common recommendations for dietary P, such as those outlined by the National Research Council (17.5 g of P/day over the entire year for cows weighing 450 kg), exceed the basic requirements for beef cattle; 12 g of P/day is adequate for 450-kg beef cows.

In vivo and in vitro inhibition of human histidine decarboxylase by (S)-alpha-fluoromethylhistidine.

Histidine decarboxylase (HDC) activity in Ficoll-Hypaque purified human peripheral blood leukocytes (PBL) was determined by measuring the formation of [3H]histamine from L-[3H]histidine. HDC activity was inhibited in vitro to more than 90% by (S)-alpha-fluoromethylhistidine (alpha-FMH) at concentrations of 10(-5) M and above. Both polymorphonuclear and mononuclear cells possessed HDC activity, but on a per cell basis the former had several-fold higher enzyme activity than the latter. In safety and tolerability studies, alpha-FMH was administered orally to healthy human subjects twice daily for 7 days at doses of 2.5, 10, 50 and 100 mg per person. A dose-dependent inhibition of HDC activity was observed in PBL that were isolated both at 12 hr after administration of the first dose of alpha-FMH and after treatment for 1 week. At the 50 and 100 mg doses of alpha-FMH, there was complete inhibition of HDC activity and partial inhibition at the 10 mg dose. Twenty-four hours after the last dose, HDC activity had recovered to 64-100%, 44-46%, and 30-52% of control values in subjects that received 10, 50 and 100 mg alpha-FMH respectively.

Attaining and measuring physical fitness in dairy cattle.

Research with dairy cows was to determine whether dairy cows in confinement are in poor physical condition, if programmed exercise improves physical fitness, and which physiological measures are most useful indicators of physical fitness. If maintenance of physiologic homeostasis when an animal is under a work load is a valid criterion of physical fitness, dairy cows in confinement are in poor physical condition, and physical fitness can be improved by programmed exercise. Walking cows at 4 km/h for a distance of 8 km daily for 5 days per wk for 8 wk prepartum improved their physical fitness. Useful hemic indicators of improved fitness were less increase in erythrocyte numbers and hemoglobin concentration and less decrease in oxygen tension, carbon dioxide, carbonic acid contents, and base excess than in poorly conditioned cattle under work load. Venous blood was satisfactory for these measurements. Also valued were heart and respiratory rates, except these rates being sensitive to extraneous disturbances may be less reliable.

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. II. Determinant specific suppression of in vitro T cell responsiveness parallels a selective suppression of anti-hapten but not anti-carrier antibody responses.

Using an antigen of defined physical structure with precisely mapped immunogenic sites, we asked whether those molecular sites previously shown to be critical for immune response gene-mediated initiation of T cell proliferation and T help correspond to the same molecular regions capable of inducing antigen-specific suppression of T cell proliferation and antibody production. Inbred strain 2, 13, and 2 x 13 F1 hybrid guinea pigs were immunized with various species variants or fragments of insulin adjuvant before subsequent immunization with antigen in complete Freund's adjuvant. Analysis of the patterns of depressed T cell responsiveness showed a striking correspondence to the Ir gene-dependent mechanism that controls the recognition of discrete regions within the insulin molecule observed in T cell help in antibody production. In addition, suppression of carrier-specific T cells parallels suppression of anti-hapten antibody responses when hapten is presented on the suppressed carrier without a concomitant suppression of the anti-carrier antibody response.

Macrophage-lymphocyte interaction and genetic control of immune responsiveness.

We have reviewed briefly some of the diverse functions of macrophages in the immune response. Clearly, this population of cells interact physically with lymphoid cells, are required for activation of T cells, and process various protein antigens. Finally, we have studied the immune response to insulin in order to unify these previous data in such a way to demonstrate the active role of macrophages in the regulation of the immune response. The function of the Ir gene in the guinea pigs appears to be an intramolecular selection of discrete regions within the antigen for recognition by the T cell. The data presented suggest that this function operates at the level of the macrophage.

Parainfluenza-3 virus exposure of beef heifers prior to natural breeding.

Ninety-six Hereford heifers (approximately 7 months of age) were randomly divided into 2 equal groups and housed 1.6 km apart (with 2 replications in time, 1 year apart). At 15 months of age, 1 group/replicate was inoculated with parainfluenza-3 virus, and the other group was given virus-free spent culture medium. Twenty-four hours later, 2 virgin bulls (2 years old) were placed with each group (24 cows) for natural breeding. Viral inoculation caused a twofold increase in parainfluenza-3 titer and a 0.3 C body temperature increase. There was no effect recognized from the virus on natural breeding efficiency.

Genetic control of the immune response to insulin: its dependence upon a macrophage mediated selection of distinct antigenic sites.

The immune response to insulin, in both mouse and guinea pig , is under control of H-linked immune response genes. When immunized with either pork or beef insulin to CFA, both strain 2 and 13 guinea pigs respond by antigen-specific lymphocyte proliferation and synthesis of specific antibody. The specificity of the elicited antibodies are indistinguishable between these inbred strains. By contrast, strain 2 T cells recognize a distinct region of the A chain alpha loop consisting of amino acids residues 8, 9 and 10, while strain 13 T cells see an as yet undefined region of the B chain. H2b (A chain alpha loop responder) and H2d (B chain responder) mice similarly discriminate which area of the molecule are recognized by their T lymphocytes. The function of the Ir gene, in both the guinea pig and mouse appears to be an intramolecular selection of discrete regions within the antigen for recognition by the T cell. The data presented suggest that this function operates at the level of the macrophage.