Lysosomal enzyme secretion by macrophages during intracellular storage of particles.

Cultured mouse peritoneal macrophages containing previously endocytosed zymosan or small-fibre asbestos (but not latex or sucrose) were shown to release selectively into the medium the lysosomal hydrolase beta-N-acetylglucosaminidase. Thus macrophage lysosomal enzyem secretion was experimentally dissociated from endocytosis (as the residual external particles were washed away from the cells). The cells remained viable, and total activities of both N-acetyl-beta-D-glucosaminidase and of lactate dehydrogenase (a cytosol enzyme) rose with time. The relevance of such secretion by macrophages containing stored materials to chronic inflammatory processes is discussed.

Description and validation of an in situ autoperfusion method to determine nutrient absorption and metabolism in bovine small intestine.

Holstein bull calves, 8 to 12 wk of age, were anesthetized with halothane gas. An approximate 20-cm section of small intestine, 60 to 90 cm proximal to the ileocecal junction was clamped to isolate blood circulation to a single set of arcuate vessels and to form an intestinal segment fitted for infusion and drainage. The vein was catheterized to allow total venous collection. Donor blood was transfused via jugular vein to replace venous drainage. This technique was evaluated in four calves by exposing the lumen to eight replications (12 or 20 min incubation, 30-min wash with 39 C saline) of 16 mM L-Met (14C-labeled). Time course appearance of Met in venous blood indicated similar rates and patterns of absorption for individual calves. There were no clinically significant alterations in jugular blood chemistry profiles across replications. Four calves were used to evaluate the effect of three isotonic perfusion media (saline, Krebs-Ringer bicarbonate and M-199 tissue culture media) on Lys and Met absorption. Venous flow rates and absorption of Lys were faster with Krebs buffer than with other media. Perfusate medium did not influence venous flow rates or absorption of Met. Effect of restricting venous flow on absorption of Lys and Met was evaluated in two calves. Flow was alternately controlled (6.5 ml/min) or allowed to flow freely (mean = 12.2 ml/min). Restricting flow decreased steady-state absorption. Light and scanning microscopy indicated maintenance of mucosal tissue integrity throughout 8 h of anesthesia. Results demonstrate validity of the in situ technique to study nutrient absorption in the young bovine.

Effect of intestinal blood flow on absorptive site blood flow and lysine absorption as examined in situ in Holstein calves.

Total blood flow from an intestinal segment (TBF) was altered to determine effects on blood flow at the absorptive site (ASBF) and lysine absorption. Venous blood flow was restricted using a peristaltic pump to 20, 35, 50, 65, and 80% of the initial unrestricted rate. Lysine absorption and ASBF were determined from recovery of 14C and 3H in blood from intestinally perfused [14C]L-lysine and 3H2O, respectively. Fluid flux in the intestinal lumen was estimated from the difference in polyethylene glycol concentrations in luminal infusate and effluent. Restriction of TBF proportionally reduced ASBF, which composed 3 to 6% of TBF. Lysine absorption was reduced linearly during reduction of TBF. Fluid absorption varied among calves but was independent of TBF. Differences between loss of radioactive marker from perfusate and recovery in blood suggested a loss of 3H2O from the intestinal segment that was independent of TBF. Changes in blood flow to the small intestine may affect nutrient absorption in ruminants.

Congenital urethral obstruction, uroperitoneum, and omphalitis in a calf.

A neonatal calf with anuria, uroperitoneum, azotemia, hyperphosphatemia, and hyperkalemia was found to have a congenital urethral obstruction. The calf had a concomitant infection of the internal umbilical remnants. Surgical intervention corrected both conditions.

Intestinal strangulation associated with omphaloarteritis in a calf.

In a 1-month-old calf, intestinal strangulation developed when a jejunal segment wrapped around a bandlike adhesion between the greater omentum and an abscess of the left umbilical artery. Signs included abdominal distention, cessation of defecation, and purulent inflammation of the external umbilical stalk. Despite vigorous care, the calf died of shock 4 hours after admission.

Varying protein content and nitrogen solubility for pluriparous, lactating Holstein cows: lactation performance and profitability.

Effects on total lactation performance of varying ration crude protein (15.3 vs. 13.6% of dry matter) and nitrogen solubility (35 vs. 45% of total nitrogen) in early lactation was studied using 57 pluriparous Holstein cows. Grain was fed according to production so as to minimize change in body weight throughout lactation. Forages high in nitrogen solubility, corn and grass silages, were fed free-choice. Percent concentrate in ration dry matter was highest (64%) 5 to 8 wk postpartum and lowest (3%) 33 to 44 wk postpartum. Protein and nitrogen solubility were varied by formulating four protein supplements fed as 10% of the grain allocation, so differences in treatments applied narrowed as lactation progressed. Cows fed the medium-protein diets produced 196 kg more milk than those receiving low-protein diets, but their peak daily milk yield was only .6 kg higher. Cows receiving rations with reduced nitrogen solubility produced 347 kg more milk than those fed the higher solubility diets, but their peak daily milk yield was 1.0 kg lower. Income above fed cost for the lactation was highest and postpartum loss in body weight was least for cows receiving medium-protein and low-solubility rations in early lactation, but no differences were significant. Both milk yield (38.3 to 40.6 kg) and total dry matter intake (3.74 to 3.91% body weight) means were maximum 6 to 7 wk postpartum, but lactation performance was not proportional to peak milk yield. When cows are fed to minimize body fat mobilization, response to increased protein in the diet is small. Reducing nitrogen solubility of silage-based diets increased milk produced per unit grain fed. There were no adverse effects of treatments on breeding efficiency or herd health.

Activation of IL-6 production by UV irradiation of blood mononuclear cells from patients with systemic lupus erythematosus.

Cultured mononuclear cells from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and normal donors were assayed for their ability to secrete IL-6 both spontaneously and after exposure to UV light. Mononuclear cells from SLE, RA and atopic control patients produced IL-6 spontaneously, while those from normal donors did not. Spontaneous production of IL-6 occurred in the non-adherent cell population. UV light-induced IL-6 production was confined exclusively to the SLE patients and was present only in the macrophage/monocyte fraction. This stimulation was induced by wavelengths in the UVA, UVB but not the UVC portion of the spectrum. These results suggest that cytokine release may be involved in the exacerbations of SLE provoked by photosensitivity.

Development of rumen function in calves: nature of protein reaching the abomasum.

Effects of weaning age (4 and 8 wk) and ration (complete pelleted starter and unpelleted starter plus alfalfa-grass hay) on development of ruminal function were tested in a split-plot design. Maturity of ruminal function was estimated by the contribution of bacterial nitrogen to total nitrogen reaching the abomasum, essential amino acid composition of bacterial and abomasal protein, and ruminal volatile fatty acid concentrations. Sixteen Holstein bull calves were fitted with rumen and abomasal cannulas by 1 wk of age, and ingesta were sampled twice weekly from 2 to 11 wk of age. Contribution of bacterial nitrogen to total nitrogen in abomasal contents was similar to that of mature ruminants by 5 and 7 wk of age for calves weaned at 4 and 8 wk of age, respectively. Concentrations of ruminal volatile fatty acids indicative of mature ruminal function were reached by 5 wk of age. Pattern of essential amino acids in bacterial cells of the rumen was not affected by age, weaning age, or ration and was similar to that of mature ruminants. Analysis of abomasal digesta indicated no effect of starter ration and no effect of age or weaning age on the relative proportion of essential amino acids except lysine and arginine. Lysine decreased and arginine increased linearly from 2 wk until weaning.

2',5'-oligoadenylate synthetase induction in lymphocytes of patients with connective tissue diseases.

Spontaneous production of the interferon induced enzyme 2',5'-oligoadenylate synthetase (2',5'-A synthetase) was significantly greater in blood lymphocytes of patients with systemic lupus erythematosus (SLE), Behçet's syndrome, and rheumatoid arthritis than in control lymphocytes. Alpha interferon (IFN-alpha) induced comparable production of enzyme in normal, rheumatoid, and Behçet's lymphocytes, and reduced, but still appreciable amounts in SLE lymphocytes. Phytohaemagglutinin (PHA) did not induce enzyme. Thus spontaneous production of 2',5'-A synthetase is encountered in several connective tissue diseases and seems likely to be induced by in-vivo exposure to interferons.

Selective immunosuppressive effects of measles virus infection.

The antibody response to diphtheria toxoid by cultured tonsil cells was suppressed by measles virus, which productively infected a small percentage of both T and B lymphocytes. However, infection of cultures spontaneously secreting antibody and immunoglobulin (with Herpes simplex virus) or measles virus did not result in immunosuppression. Immunosuppression by measles virus may thus be attributed to a selective effect on the inductive phase of the response and suggest that infection of immunoglobulin-producing B lymphocytes does not alter the ability of these cells to secrete antibody.

Induction of macrophage lysosomal enzyme secretion by agents acting at the plasma membrane.

Cultured mouse peritoneal macrophages were shown to secrete the lysosomal enzyme N-acetyl-glucosaminidase (N-ac-Glu) in response to IgG-Sepharose and some other non-endocytosable particles without substantial release of the cytoplasmic enzyme, lactate dehydrogenase. The interaction with IgG-Sepharose was studied in some detail, and was shown to be time- and dose-dependent, and to leave the cells viable. Ovalbumin and glycine insolubilised on Sepharose did not induce secretion. By means of several control experiments, it was demonstrated that the IgG-Sepharose exerted its effects directly on the plasma membrane. Thus, normal macrophages can secrete in response to certain agents which act solely on the plasma membrane. This mechanism of induction of secretion is probably quite distinct from those previously established, which involve secretion during phagocytosis, during intracellular storage of phagocytosed materials or during pharmacological intervention by cytochalasin B.

Herpes simplex virus and the rheumatic diseases.

A viral aetiology for rheumatoid arthritis and inflammatory connective tissue diseases has been sought in general terms first, by studying viral growth patterns in lymphocytes from the blood and lesions of patients affected second, by analysing lymphocyte concentrations of the interferon-induced enzyme 2-5 oligo-adenylate synthetase (2-5 A); and third, by probing Southern blots of lymphocyte DNA with viral probes. Indirect evidence consistent with a viral aetiology has been found in several such diseases, but direct proof has been difficult to adduce. There is some suggestion that herpes simplex viral (HSV) DNA is present in Behcet's blood lymphocytes, but the findings are inconsistent. It is also plausible that viruses such as HSV do not induce these diseases through classic immunopathological mechanisms, but as "promoters" of abnormal lymphoproliferation in individuals with predisposing defects, possibly related to selective DNA repair defects.

Methionine deficiency in early-weaned dairy calves fed pelleted rations based on corn and alfalfa or corn and soybean proteins.

Holstein bull calves weaned at 4 wk of age were assigned to one of three replicated 4 X 4 Latin squares at 5 to 6 wk. Trials were abomasal infusion of 0, .30, .60, and either 90 (Trials 2 and 3) or 1.20 g (Trial 1) L-methionine/kg pelleted starter ration consumed. In Trials 1 and 2, ration ingredients of the ration contributed the following percentages of total crude protein: corn meal 39, alfalfa meal 48, and soybean meal 12; dry matter of rations contained 13.6 and 13.9% crude protein and .26 and .32% sulfur. Infusion of .60 g methionine in Trial 1 tended to depress excretion of nitrogen in urine and increase nitrogen retention. In Trial 2, urine nitrogen excretion and retained nitrogen responded in a quadratic fashion; infusion of .30 and .60, and either .90 (Trials 2 and 3) or 1.20 g (Trial 1) L-methionine/kg pelleted starter ration consumed. In Trials 1 and 2, ingredients of the ration contributed the following percentages of total crude protein: corn meal 39, alfalfa meal 48, and soybean meal 12; dry matter of rations contained 13.6 and 13.9% crude protein and .26 and .32% sulfur. Infusion of .60 g methionine in cretion 9%. Relationship between plasma methionine and infused methionine was linear for all trials. Methionine is the first limiting amino acid for early-weaned calves fed complete pelleted rations based on corn and either alfalfa or soybean proteins.

The selective release of lysosomal acid hydrolases from mouse peritoneal macrophages by stimuli of chronic inflammation.

The paralelism between the capacity of various agents to elicit chronic inflammatory responses in vivo and to induce the selective release of lysosomal enzymes from cultures of mouse peritoneal macrophages in vitro is discussed. Zymosan elicits an intense inflammatory response when injected i.m. in mice. Chrysotile asbestos produces a response of a similar nature and intensity as is seen with zymosan, while injections of acid-leached asbestos and polystyrene latex are not followed by inflammation. It is also shown that zymosan and asbestos induce a dose-dependent increase in the total enzyme activity of an inflamed muscle. On the other hand latex and acid-leached asbestos caused no significant increases in lysosomal enzyme levels. Agents eliciting inflammatory responses, such as zymosan and chrysotile asbestos induce a selective release of acid hydrolases from cultured macrophages; in contrast agents lacking the capacity to induce inflammation, such as latex and acid-leached chrysotile asbestos, do not induce the release of lysosomal enzymes from cultured macrophages.

Response to abomasal infusion of methionine of weaned dairy calves fed a complete pelleted starter ration based on by-product feeds.

Three abomasal infusion trials were to determine the first-limiting amino acid for nitrogen retention of postweaned Holstein bull calves fed a starter ration based on cereal by-product feeds. Ration ingredients contributed the following percentages of total amino acids: wheat middlings 38, brewer's grains 24, hominy feed 21, oat hulls 10, distiller's grains 6, and molasses 1. Dry matter of the pelleted ration contained 13 to 14% crude protein and .23 to .26% sulfur. Each trial was a replicated 4 x 4 Latin square with 9-day periods. Calves were weaned at 4 wk and infusions initiated at 5 to 6 wk of age. Abomasal infusion of the 10 essential amino acids, an isonitrogenous quantity of an equimolar mixture of alanine, asparagine, glutamate, glycine, and serine, or an isocaloric quantity of glucose indicated that essential amino acid(s) rather than nonspecific nitrogen or energy was limiting protein synthesis. Quadratic responses in urine nitrogen excretion and retained nitrogen occurred when 0, .3, .6, and 1.2 g L-methionine/kg starter ration were infused. Greatest responses occurred with .6 g methionine, which increased retained nitrogen 11 and 19% and decreased urine nitrogen 7 and 10%. The relationship between infused methionine and plasma methionine was linear. Infusions in all trials had no effect on digestibility of energy and nitrogen.

Effects of immunosuppression on herpes simplex virus growth in lymphocytes of patients with connective tissue diseases.

The growth of herpes simplex virus (HSV) was measured in blood lymphocytes isolated from patients with connective tissue diseases receiving immunosuppressive drugs. Whereas lymphocytes from such patients were frequently nonpermissive for HSV before treatment, they became permissive in most patients after treatment. The exception was the persistence of nonpermissiveness in lymphocytes of treated patients whose disease was associated with malignant disease.

Cytokine-independent progression of immunoglobulin production in vitro by B lymphocytes from patients with systemic lupus erythematosus.

B lymphocytes from patients with systemic lupus erythematosus (SLE) secreted high levels of immunoglobulin spontaneously when cultured in vitro. Addition of the cytokines interleukin-2, interleukin-4 and interleukin-6 either alone or in combination failed to augment spontaneous immunoglobulin synthesis. Percoll-separated low-density SLE B lymphocytes matured into immunoglobulin-secreting cells also independent of exogenous interleukins. During maturation these cells became enlarged and less dense, and began to express CD23. This was in contrast to normal B cells, which did not secrete immunoglobulin spontaneously but synthesized IgM after interleukin stimulation. These results indicate that in vitro immunoglobulin synthesis by SLE B cells is already initiated in these cells and progresses independently of further stimulatory manoeuvres.

Defective repair of 0(6)-methylguanine in autoimmune diseases.

On the premise that somatic mutations may be involved in the pathogenesis of diseases such as systemic lupus erythematosus and rheumatoid arthritis, the proficiency of repair of 0(6)-methylguanine, a powerful, premutagenic, directly miscoding base lesion, was examined in the DNA of peripheral-blood mononuclear cells (mainly lymphocytes) from patients with such diseases. The capacity of lymphocytes to repair this lesion was impaired in many patients with autoimmune disease. In some healthy controls repair was also defective whereas in other, nonautoimmune diseases, it was not. These findings support the hypothesis that this defect of repair was not simply a result of the disease state. It is therefore postulated that defective repair of 0(6)-methylguanine could be one of the factors determining the susceptibility of autoimmune diseases in genetically predisposed individuals.

Increased sensitivity of lymphocytes from patients with systemic autoimmune diseases to DNA alkylation by the methylating carcinogen N-methyl-N-nitrosourea.

Lymphocytes from patients with various diseases associated with autoimmunity showed both impaired capacity to repair O6-methylguanine (a powerful, promutagenic, directly miscoding base lesion) and increased sensitivity to the cytocidal effects of cellular methylation by N-methyl-N-nitrosourea (MNU) compared with normal controls and patients with other disorders. Defective repair of O6-methylguanine was significantly associated with arthritis and myositis in the group with systemic lupus erythematosus (SLE), and increased sensitivity to the toxic action of MNU was associated with the presence of immune complexes and the administration of steroids to patients with Behçet's syndrome. The results indicate that lymphocytes from patients with the autoimmune diseases studied are more susceptible to DNA damage with possible relevance to aetiopathogenesis.