Reversal of virus-induced immune suppression

We studied the immunosuppressive capacity of splenic lymphocytes from rabbits at different stages of progressive myxosarcoma induced by malignant rabbit fibroma virus (MV). Spleen cells taken from rabbits 7 days after virus inoculation proliferate poorly in response to Con A, and suppress normal responses to the mitogen. Those from animals 11 days after virus injection have recovered partially from MV-induced suppression. Further, their Con A responses are no longer suppressed by day 7 spleen cells. Supernatants from cultures of spleen cells from rabbits given MV 7 days previously suppress both antibody-producing and proliferative responses to unrelated antigens. Comparable supernatants from rabbits receiving MV 11 days before sacrifice neither suppress nor augment such responses. Mixing cells from 7 or 11 day MV rabbits with normal spleen cells gives similar results. When supernatants from spleen cell of rabbits with tumors induced 7 and 11 days previously are mixed, the supernatants from rabbits with 11-day-old tumors inhibit the suppressive capacity of those from animals with 7-day-old tumors. Similarly, mixing spleen cells from rabbits given MV 7 and 11 days previously results in culture supernatants that do not suppress normal antibody and proliferative responses. The ability of cells from rabbits given MV 11 days before to inhibit the effects of cells from rabbits given MV 7 days previously does not involve the production of interferon. Thus, despite progressive tumor burden, immunologic recovery is observed in rabbits 11 days after tumor virus inoculation. One factor in this recovery may be the generation of active inhibitors of virus-induced immunosuppression. Similar mechanisms may apply to recovery of immunologic function in other virus infections as well.     

Production of macrophage migration inhibitory factor(s) (MIF) by virus-transformed cells

Rodent cell lines transformed by SV40, polyoma virus and Rous sarcoma virus cultured in vitro release material into the culture medium which inhibits the migration of guinea pig macrophages. Similar macrophage migration inhibitory factors (MIF) were not detected in cell-free supernatants harvested from untransformed cell cultures. Comparison of the MIF produced by virus-transformed cells with MIF derived from peripheral lymphocytes stimulated in vitro by phytohemagglutinin (PHA) revealed that they had similar molecular weights (25 000), heat stability and were both inhibited by α-fucose and lotus agglutinin. Incubation of MIF-containing cell-free supernatants from transformed cells with pancreatic trypsin inhibitor, soybean trypsin inhibitor and diisopropylfluorophosphate eliminated the MIF activity. The possible identity of the MIF released by transformed cells as a protease is discussed with reference to a potential role in modifying the surface properties of transformed cells.     

Brugia malayi antigens associated with lymphocyte activation in filariasis

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.     

Trichinella spiralis: Generation in the presence of rat serum of factors chemotactic for rat cells

Chemotaxis of rat peritoneal cells, of which the eosinophil was the predominant migratory cell type, toward incubates of Trichinella spiralis was studied using a modified Boyden chamber. Excysted muscle larvae, preadults, and adults were incubated in a buffered medium for 20 hr at 37 C. Worms were incubated alone or with serum or spleen cells, or both, from immune and nonimmune rats. Incubates of worm stages alone possessed no chemotactic activity as compared with incubation medium as a negative control and zymosan-activated serum as a positive control. Both normal and immune sera tested alone stimulated cell migration to the same degree. Incubates of spleen cells from either normal or immunized hosts did not show chemotactic activity. Chemotaxis caused by normal and immune sera were not altered by incubation with homologous spleen cells. Addition of larva, preadults, and adult worms to sera, however, enhanced chemotactic activity over sera alone. Chemotaxis caused by larvae plus immune sera was significantly greater than that stimulated by larvae plus normal sera. This difference decreased when preadults were substituted for larvae and was not observed when adult worms were used. Reversal of the chemical gradients showed that active cell migration caused by various incubates was due to Chemotaxis.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of mononuclear cells infiltrating into tumors. V. A. soluble factor involved in the regulation of cytotoxic/suppressor T cell infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of cytotoxic/suppressor T lymphocytes in tumor tissues. We found that tumor-infiltrating helper/inducer T cells isolated from T-9 gliosarcoma-sensitized rats between 4 and 6 days after T-9 gliosarcoma inoculation produced a lymphocyte migration factor (LMF) during in vitro culture. Four peaks of LMF activity (A through D) were detected upon fractionation of LMF by using a Mono Q anion exchange column chromatography. Peak C exhibited the strongest activity among the four peaks of LMF. The action of peak C was chemotactic, but not chemokinetic. Peak C had an isoelectric point of 8.0 and a Mr of 26,000 Da. Only cytotoxic/suppressor T cells were found to be sensitive to peak C in vitro as well as in vivo. It is thus likely that peak C is responsible for the infiltration of cytotoxic/suppressor T cells into tumor tissues. The infiltration of lymphocytes into tumor tissues might also be regulated by the expression of lymphocyte sensitivity for LMF. The target molecule for LMF at 4 days may involve an asparagine-linked oligosaccharide.     

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.     

Effect of ischemia-reperfusion injury on the morphology of peroxisomes

We have previously demonstrated that ischemic injury changed the density of peroxisomes into two distinct peaks, one with a normal density (1.21 g/cm³; Peak I) and a second peak with a lighter density (1. 14 g/cm³; Peak II).We studied the peroxisomes from both peaks under the Electron microscope. Examination of peak I following ischemia showed loss of matrix proteins and damaged limiting membranes with leakage of DAB positive material in direct proportion to the duration of ischemia. Upon reperfusion of the ischemic liver Peak I showed more severe damage to the organelle. These observations clearly demonstrated that ischemia reperfusion injury causes structural damage to peroxisomes. Interestingly ultrastructural examination of Peak II following ischemia showed evidence of perisomal proliferation with budding of existing peroxisomes and the presence of micro peroxisomes (changes similar to those noted under conditions leading to perisomal proliferation). However, peak II following reperfusion showed only damaged organelle. These observations underline the importance of peroxisomes in the response of the cell to ischemia-reperfusion injury.     

Existence in fetal and adult rat of several forms of material reacting with anti-insulin antibody (IRI).

The double antibody procedure detects 3 peaks in the elution fractions of adult or fetal rat sera, after passage on Sephadex G50 or G100 columns. Peak A (apparent MW 6000) contains insulin monomer; peak B (apparent MW 10-12000) is tentatively attributed to proinsulin (or proinsulin like substances); peak C (apparent MW 50-100000) is similar to the so called "big big" insulin. During intravenously induced hyperglycemia, the 3 peaks show parallel increases, but, after the disappearance of peaks A and B in streptozotocin treated rats, peak C remains unaltered. Pancreatic extracts and secreta present a very minor and inconstant peak C, the bulk of their immunoreactive material belonging to peaks A and B. A companion paper further discusses the nature of peaks B and C materials.     

Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.     

S_(180)肉瘤细胞对小鼠基因组DNA结构变化影响的核磁共振氢谱~1H—NMR研究

本实验利用核磁共振技术,对体内培养了腹水癌小鼠肝细胞基因组DNA的结构变化进行了测试,发现随致癌时间的延长,基因组DNA内胸腺嘧啶核苷酸周围的化学环境发生了定向的变化,造成DNA核磁共振氢谱中,部分胸腺嘧啶的甲基质子发生了化学位移,在′H-NMR图谱中1.9ppm峰Ⅰ减小,而2.6-2.7ppm区域的峰Ⅱ却明显增加,结果使双峰比(峰Ⅰ积分面积/峰Ⅱ积分面积)显著下降,本文为研究癌症发生和发展的遗传学机制提供了一个新的方法.     

Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.     

Demonstration of active suppressor cells in spleens of young NZB mice

NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.     

L-fucose blocks MIF/MAF priming of rabbit alveolar macrophages for a PMA-induced oxidative response

The effect of L-fucose on the priming of AM from normal adult rabbits and their subsequent chemiluminescent (CL) responses to phorbol myristate acetate (PMA) was investigated. It was observed that 12.5 mM L-fucose, but not D-fucose, blocked the "spontaneous" priming of normal AM during an 18-hr incubation period in serum-free RPMI 1640 medium by about 40% (P less than 0.01) as detected by their CL responses to a PMA challenge. In addition, the optimal concentration of L-fucose (12.5 mM) blocked MIF/MAF priming during the 3- or 18-hr incubation period by 71 or 93% (P less than 0.05), respectively, as determined by their CL responses following PMA challenge. It is of particular significance that D-fucose was inactive. These results, together with previously published data, indicate that L-fucose (a) blocks priming of AM for an oxidative response, (b) stimulates random migration of AM, and (c) reverses migration inhibition produced by migration inhibition factor.     

Interleukin 1 production and accessory cell function of rat alveolar macrophages exposed to mineral dust particles

Immunostimulatory effects of respirable mineral dust particles on alveolar macrophages (AM) and T lymphocytes were tested in vitro. When rat AM were incubated with fibrogenic silica and asbestos particles, a significant interleukin 1 (IL-1) activity was generated into the culture supernatants, whereas neither AM alone nor AM incubated with non-fibrogenic titanium dioxide (TiO2) particles, however, produced a detectable amount of IL-1. Interleukin 2 (IL-2) activity, as tested with IL-2-dependent CTLL-2 assays, was not detectable from all of these cultures. It was also revealed that concanavalin A-induced proliferation of T lymphocytes was enhanced in autologous AM and nylon wool-passed spleen cell co-cultures incubated only with fibrogenic particles, but not with non-fibrogenic particles. Higher IL-1 activity was detected only from co-cultures exposed to fibrogenic particles, whereas IL-2 activity was almost similar among these co-cultures. These results indicate the differences in immunostimulatory effects on pulmonary macrophages and T lymphocytes among a variety of mineral dust particles.     

Unique properties of cluster of differentiation 93 in the umbilical cord blood of neonates

It has previously been reported by these authors that cluster of differentiation (CD) 93 is co‐expressed on naive T‐lymphocytes (CD4⁺CD45RA⁺ cells) in neonatal umbilical cord blood cells (UCBCs) but not on normal adult peripheral blood cells (PBCs). In this study, expression of CD93 on other lymphocyte subsets and the concentration of soluble formed CD93 (sCD93) in serum or culture supernatants from neonatal umbilical cord blood (UCB) was examined. It was found that CD93 is also co‐expressed on CD2⁺, CD16⁺, CD56⁺ or CD25⁺ cells in the lymphocyte population of neonatal UCBCs, but not on normal adult PBCs. The concentrations of sCD93 in serum and culture supernatants from neonatal UCB were significantly greater than those from normal adult peripheral blood. The concentrations of sCD93 in culture supernatants from neonatal UCBCs and normal adult PBCs treated with phorbol 12‐myristate 13‐acetate (PMA) were significantly enhanced compared with those without PMA treatment. The degree of enhancement of sCD93 by PMA in culture supernatants from neonatal UCBCs was significantly greater than that of normal adult PBCs and enhancement of sCD93 by PMA in the culture supernatants from neonatal UCBCs and normal adult PBCs was significantly suppressed by PKC inhibitor. Interestingly, the high concentration of serum sCD93 in neonates was significantly decreased in sera from infants at 1 month after birth. Expression of CD93 on the lymphocyte population of PBCs from infants at 1 month after birth was also significantly decreased, compared with that for neonatal UCBCs. These findings indicate that CD93 in neonatal UCB has unique properties as an immunological biomarker.     

Eimeria tenella and E. acervulina: lymphokines secreted by an avian T cell lymphoma or by sporozoite-stimulated immune T lymphocytes protect chickens against avian coccidiosis

The role of avian lymphokines as nonspecific immunomodulators of host immunity against the intracellular parasite Eimeria was investigated. Prophylactic treatment of normal chickens with crude cell-free supernatants obtained from JMV-1 culture, concanavalin A (Con A)-stimulated normal spleen cells, or sporozoite-stimulated immune T cells prior to inoculation with E. tenella or E. acervulina conferred significant protection. These crude cell-free culture supernatants also inhibited intracellular development of eimerian parasites in vitro. Avian macrophages pretreated with these supernatant preparations showed inhibitory activity against Eimeria. This inhibitory activity could not be ascribed to anti-Eimeria antibody, complement, or cell-free Marek's disease virus and was therefore considered to be due to immunomodulating lymphokines present in the culture supernatants. These results suggest that JMV-1-transformed T lymphoblastoid cells, immune T lymphocytes, and Con A-stimulated normal spleen cells secrete lymphokines that can enhance host immunity in a nonspecific manner and implicate cell-mediated immunity as a major mechanism of the protective host immune response against eimerian infections.     

The mechanism of action of soluble lymphocyte mediators. IV. Effect of migratio inhibitory factor (MIF) on macrophage cyclic AMP and on responsiveness to adenylate cyclase stimulators.

Intracellular levels of cyclic 3′,5′-adenosine monophosphate (cAMP) in purified guinea pig peritoneal macrophages were elevated following incubation with the adenylate cyclase stimulators prostaglandins E₁ and E₂ (PGE₁, PGE₂), isoproterenol, and cholera toxin. Exposure of macrophages to antigen-stimulated lymphocyte culture supernatants, containing migration inhibitory factor (MIF), resulted in a moderate but consistent decrease in the cAMP level, which was best expressed after 1–2 hr of incubation. Incubation of macrophages with MIF-containing supernatants or partially purified MIF for 1–2 hr resulted in reduced cAMP accumulation in response to PGE₁, PGE₂, isoproterenol, and cholera toxin (nonspecific refractoriness). These findings indicate that MIF-induced inhibition of macrophage migration is not due to an increase in the cellular level of cAMP and that the reduction in cAMP concentration, caused by MIF, is probably a secondary phenomenon unrelated to the inhibition of cellular motility.     

Increased transglutaminase in the aortas of cholesterol-fed rabbits: occurrence of buffer soluble and insoluble forms and an inhibitor.

Rabbits were fed for 10-12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl-sucrose-dithiothreitol-Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.