Lymphocyte-defined loci in cattle.

Using the results of all paired one-way mixed lymphocyte culture tests on families of half-sibs, we have established that the lymphocyte-defined system in cattle contains a minimum of two loci. The methodology presented is applicable to studies of the lymphocyte-defined systems of other species.

CTGF, intestinal stellate cells and carcinoid fibrogenesis.

AIM: To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell. METHODS: GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay. RESULTS: Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (> 2-fold, P < 0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (> 3-fold, P < 0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P < 0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P < 0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P < 0.05) compared to non-fibrotic GI carcinoids (< 15 ng/mL). CONCLUSION: SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma.

BACKGROUND: Over-expression of connective tissue growth factor (CTGF) is a hallmark of fibrotic disease, including scleroderma. CTGF acts with the pro-fibrotic cytokine TGFbeta to promote sustained fibrotic responses in vivo. Elevated production of CTGF might be responsible for maintenance of the fibrotic phenotype in scleroderma. Assays of CTGF or of its fragments are potential non-invasive measures of the fibrotic response in scleroderma. AIM: To determine the utility of whole, N-terminal, and C-terminal CTGF as surrogate markers for fibrosis in scleroderma. DESIGN: Cross-sectional controlled study. METHODS: Plasma was collected prospectively from 47 scleroderma patients (26 diffuse scleroderma, 21 limited scleroderma) and 18 healthy controls. At the same time, dermal interstitial fluid was derived by a suction blister technique from the lesional skin of scleroderma patients, and from the forearm skin of healthy controls. Whole, N-terminal, and C-terminal CTGF were assayed by ELISA, using monoclonal antibodies specific for N- and C-terminal epitopes. RESULTS: N-terminal cleavage products of CTGF were present at elevated levels in the plasma and dermal interstitial fluid of scleroderma patients, compared to healthy controls. N-terminal CTGF levels in plasma and dermal interstitial fluid correlated with severity of skin disease and (negatively) with disease duration. Whole and C-terminal CTGF levels were low in blister fluid and plasma levels were not elevated in disease. DISCUSSION: These results support a role for CTGF in scleroderma-associated fibrosis and the utility of N-terminal CTGF as a marker of fibrosis.

Two molecularly independent surface receptors identify bovine T lymphocytes.

E rosette formation is a commonly used technique to identify T lymphocytes in many species; however, treatment of bovine E rosettes with rhodamine-conjugated F(ab')2 anti-immunoglobulin resulted in 10% (range 3-18%) of these cells exhibiting fluorescence. The failure of E rosettes to identify only non-immunoglobulin bearing cells suggested that an alternative method for T lymphocyte identification was essential. Using a labeled heterologous T cell antiserum and peanut agglutinin (PNA), identical populations of bovine T lymphocytes were identified. Unique and molecularly independent cell surface receptors were detected by dual fluorescent staining experiments differential inhibition of cellular binding of PNA by its carbohydrate ligand and capping experiments. Moreover, both reagents bound to approximately 62% of peripheral blood lymphocytes (PBLs) and virtually all thymocytes. Use of either T cell reagent in combination with rhodamine-conjugated F(ab')2 anti-bovine immunoglobulin provided a rapid, simple and reliable method for simultaneous enumeration of bovine T and B cells and permitted the detection of rare cells (less than 1%) expressing both T and B cell markers. Approximately 90% of all PBLs were identified as either T or B cells.

Bovine mixed leukocyte response: effect of selected parameters on cell responses.

The bovine mixed leukocyte culture (MLC) system offers potential benefit for the study of genetic and immunologic mechanisms in this species. Selected parameters of the bovine MLC have been investigated to assess their influence. The findings indicate that maximal MLC response was present at days 5 and 6 with 2 x 10(5) responder to 2 x 10(5) stimulator cells per well or greater. Serum concentrations over a wide range effectively supported cell growth. More importantly, the source and treatment of serum influenced the level of MLC response. Serum autologous to the responder which was heat treated appeared to provide maximal MLC values compared to other sources examined. Antisera to serologically-defined lymphocyte antigens of the stimulating cell population did not affect the response. One-way MLC determinations were consistent when the stimulating cell population was irradiated at 1,000 Rads or greater. Both freshly obtained lymphocytes and lymphocytes held at room temperature for 18 hr provided good sources of MLC responder and stimulator cells; however, lymphocytes held for 18 hr consistently gave higher counts and stimulation indices than freshly obtained lymphocytes. The effect of contaminating RBCs was found to enhance MLC reactivity at lower concentrations and inhibit MLC reactivity when in excess of 7 X 10(9) RBC per well. Stimulator cells were present in both macrophage enriched and depleted populations suggesting several cell populations possess antigens which lymphocytes recognize. Consideration of these parameters were essential in providing optimal, reproducible results.

A comparison of antibody responses to veterinary vaccine antigens potentiated by different adjuvants.

Six adjuvant formulations were compared for their ability to potentiate the primary and memory antibody responses in mice to three companion animal vaccine immunogens--feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and a recombinantly-derived heartworm antigen. The combination of a novel bacterial immunostimulator, gliding bacterial adjuvant (GBA), either adsorbed onto an aluminum hydroxide gel (Rehydragel HPA), or emulsified with a vehicle of polyalcohol and detergent, elicited the strongest memory responses to both virus preparations. Both forms of aluminum hydroxide gels administered without GBA gave similar levels of adjuvant effects, on par with or greater than those generated by incomplete Freund's adjuvant (IFA). The Acemannan immunostimulant was not effective in increasing the responses to the virus antigens, but increased the primary response to the heart-worm antigen over tenfold from control levels. All preparations appeared to be well tolerated, with no detectable adverse reactions observed in any of the 250 mice used. The proven safety of aluminum hydroxide adjuvants and the apparent absence of adverse reactions seen with GBA make this vehicle/adjuvant formulation worthy of additional study.

A simple method for specific depletion of B lymphocytes from bovine peripheral blood mononuclear cells.

The treatment of bovine peripheral blood mononuclear cells with rabbit anti-bovine immunoglobulin, goat anti-rabbit immunoglobulin (GAR) and complement resulted in the specific lysis of all surface immunoglobulin (SIg) bearing B lymphocytes. No SIg+ lymphocytes were detected after 12 hours of culture following lytic treatment, whereas cells treated with antibody without complement readily stained for SIg. The percentage of cells forming E-rosettes or binding peanut agglutinin (PNA) increased following lysis. The percentage of latex-ingesting monocytes also increased after lysis even though many of these cells had cytophilic Ig. B lymphocyte-depleted (T cell-enriched) populations cultured with phytohemagglutinin (PHA) and concanavalin A (Con A) were never less reactive than unseparated cells. No differences in background mitosis was observed for these 2 cells preparations. These results suggest that bovine SIg- cells do not require B lymphocytes to respond to PHA and Con A and that lysis of B lymphocytes does not alter the responsiveness of the recovered SIg- cells.

Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides.

Antibodies reactive with capsular polysaccharides are considered the principal mediators of immunity against invasive diseases caused by Streptococcus pneumoniae. In this study, we tested the hypothesis that anti-pneumococcal capsular polysaccharide (PPS) antibody avidity can influence protective efficacy. We measured the avidities of individual adult postvaccination immunoglobulin G2 (IgG2) antibodies to PPS serotypes 6B and 23F and examined the relationship between avidity and opsonophagocytic and mouse-protective activities. The avidities of PPS 6B- and PPS 23F-specific IgG2 antibodies ranged from 6 to 31 nM-1 and from 3 to 20 nM-1, respectively. We observed an inverse correlation between the magnitude of avidity and the amount of antibody required to protect mice against lethal bacteremia caused by serotype 6B pneumococci. Similarly, higher-avidity antibodies were more effective than lower-avidity antibodies in vitro in mediating complement-dependent opsonophagocytosis of both 6B and 23F pneumococci. These data suggest that in adults, PPS antibodies are sufficiently polymorphic to possess biologically significant variations in avidity. We conclude that avidity functions as an important determinant of anticapsular antibody protective efficacy against pneumococci.

Intracellular pH in unanesthetized dogs during panting.

Intracellular pH, arterial blood gases and several plasma enzymes were estimated in unanesthetized dogs during a 3-hour exposure to 30 degrees C/50% relative humidity, and 40 degrees C/50% relative humidity. No change occurred during mild heat stress, whereas during severe heat stress a profound respiratory alkalosis developed together with an increase in intracellular pH from 7.03 to 7.29. Most plasma enzymes increased by about 300% or more. In spite of extreme panting body temperature rose to 42.2 degrees C. Exposure to 40 degrees C/50% relative humidity with 4% CO2 in the climatic chamber inhibited the respiratory alkalosis and the increase of plasma enzymes. Though the panting frequency was lower the ventilatory heat dissipation was more efficient. Body temperature rose to only 39.8 degrees C. It is concluded that the intracellular buffering is not able to prevent marked changes of the intracellular pH during panting.

Mean whole body intracellular pH in unanesthetized dogs: a revised method.

An improved method of calculating the mean whole body intracellular pH (pHi) by means of DMO in unanesthetized dogs is described. The elemination of DMO from the body fluid is assumed to follow a simple exponential decay with a time constant k1. The distribution of DMO into the extracellular and intracellular water is described by an exponential function (1-exp(-k2t)). From experiments in 9 unanesthetized dogs it was found that k1 = 0.008 h-1 and k2 = 1.325 h-1. Mean pHi was 7.08 +/- 0.05 at Pa(c02) = 4.1 kPa (31 mm Hg). The average difference between arterial blood and pHi was 0.35. No statistically different pHi values were found with data from arterial or mixed venous blood. Even after 24 h the method still yields reasonable values for pHi.

Bovine con A-induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action.

Bovine Concanavalin A-induced suppressor cells were generated from lymphocytes which were non-adherent to anti-immunoglobulin coated dishes and cells possessing receptors for peanut agglutinin. Bovine lymphocytes, preincubated with 25 microgram/ml of Con A for 40-45 hr, could suppress the responses of autologous cells to the mitogens Con A, PHA and PWM as much as 90% when they were cultured together at a ratio of 1:1 (suppressor cell to responder cell) or higher. Suppressor cells were not necessary at the initiation of the mitogenic assay as they could regulate responding cells if added at 48 hr in a 72 hr assay. Allogeneic responder cells could be suppressed at the same level as autologous cells indicating a lack of genetic restriction. Macrophages were not required for suppressor cell generation because peripheral blood lymphocytes (PBL) depleted of macrophages by Sepharose G-10 columns, and subsequently incubated with Con A, could suppress autologous cells to a similar degree as unseparated PBl's. Responder cells depleted of macrophages had normal mitogen responsiveness and were suppressed indicating macrophages were not required in transmission of suppressor signals. Cell to cell contact was not required for suppression connoting a soluble factor(s) as the modulator of suppression.

Determination of mean whole body intracellular pH in unanesthetized dogs.

In order to determine mean whole body pHi in unanesthetized dogs, assumptions on the urinary and intestinal excretion of DMO, the total loss of an injected single dose of [14C] DMO, and the distribution kinetics of DMO were tested experimentally. Urinary excretion of DMO was almost negligible. The best assumption on the total loss of DMO was based on the exponential decay observed over a period of 1 to 4 weeks. The biological half-life of DMO was 5 days, the time constant being --0.14 d-1. The extracellular distribution of DMO was considered to equal that of [3H] inulin. Between 1 and 7 hours after an injection of inulin in nephrectomized dogs the distribution volume increased linearly from 16% of the body weight after 2 hours to 21% after 6 hours. Based on these experimental results, pHi was determined in 16 unanesthetized dogs. 120 min after the injection of DMO pHi was 7.05 and 430 min after the injection pHi was 7.11. It is concluded that the assumption made allow the estimation of pHi in unanesthetized dogs over a period of 1 to 7 hours.

Transitivity of response in the mixed lymphocyte culture test.

We present a model of mixed lymphocyte culture (MLC) response which assumes one specificity per locus. It also assumes that an animal A will fail to stimulate animal B if, and only if, the set of specificities possessed by A is a subset of the set of specificities in B. The last assumption implies that non-stimulation is transitive; that is, if A does not stimulate B, and B does not stimulate C, then A will not stimulate C. The inclusion of antigenic sets can be used to partially order the animals in a hierarchy. Partial ordering can detect multiple lymphocyte-defined (LD) loci with relative ease; it indicates the number of antigens present in particular individuals; and it detects exceptions to the rule of transitivity which may expose immune response genes, minor loci, or other mechanisms that affect MLC response. This analytical procedure is most useful when testing half-sib families or hybrids sharing a common parental strain. We have applied this procedure to the MLC in cattle half-sib families and found that the data strongly support the existence of at least four LD loci.

Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells.

Monoclonal antibodies were raised against an 80-kDa phosphoprotein (80K) that is phosphorylated upon stimulation of gastric acid secretion and that copurifies with the acid-forming H+-K+-ATPase isolated from stimulated tissue. These antibodies were used to demonstrate that in the gastric mucosa 80K is limited to parietal cells and not found in surface, mucous neck, or chief cells. 80K was also found in other transporting epithelia, including intestine and kidney, but was not found in brain, liver, red blood cells, or colon. Immunohistological localization of 80K in resting glands revealed a fine network, projecting from the gland lumen and anastomosing throughout the parietal cell. This network is quite similar to the staining pattern for F-actin contained in microvilli that line the apical membrane of parietal cells. Stimulation of acid secretion rearranges 80K to a more rugose pattern filling the entire cell. In stimulated cells the distribution pattern of 80K is indistinguishable from that stained with antibodies against the H+-K+-ATPase. These data strongly suggest that 80K is an apical membrane protein of the parietal cell.

The bovine major histocompatibility complex (BoLa): close linkage of the genes controlling serologically defined antigens and mixed lymphocyte reactivity.

Detection of linkage between genetic loci in cattle has been hampered by the lack of large full -sib families. A unique source of full-sib families is now available from embryo transplantation. Lymphocytes from six full-sib families, ranging in size from three to seven siblings, were tested for serologically defined BoLA antigens (BoLA-A). In addition, mixed lymphocyte reactivity (MLR) was tested between all paired combinations of cells within each family to distinguish BoLA-D specificities. Serologically identical siblings within each family were reciprocally nonreactive in MLR, and vice versa; thus, no recombinants were detected between the BoLA-A and the BoLA-D loci. Classical genetic linkage analysis revealed that these loci are significantly closer than 11.9 centimorgans.