F13A1 transglutaminase expression in human adipose tissue increases in acquired excess weight and associates with inflammatory status of adipocytes.

OBJECTIVE: F13A1/FXIII-A transglutaminase has been linked to adipogenesis in cells and to obesity in humans and mice, however, its role and associated molecular pathways in human acquired excess weight have not been explored. METHODS: We examined F13A1 expression and association to human weight gain in weight-discordant monozygotic twins (Heavy-Lean difference (ΔWeight, 16.8 kg ± 7.16 for n = 12). The twin pairs were examined for body composition (by dual-energy X-ray absorptiometry), abdominal body fat distribution (by magnetic resonance imaging), liver fat content (by magnetic resonance spectroscopy), circulating adipocytokines, leptin and adiponectin, as well as serum lipids. Affymetrix full transcriptome mRNA analysis was performed from adipose tissue and adipocyte-enriched fractions from subcutaneous abdominal adipose tissue biopsies. F13A1 differential expression between the heavy and lean co-twins was examined and its correlation transcriptome changes between co-twins were performed. RESULTS: F13A1 mRNA showed significant increase in adipose tissue (p < 0.0001) and an adipocyte-enriched fraction (p = 0.0012) of the heavier co-twin. F13A1 differential expression in adipose tissue (Heavy-Lean ΔF13A1) showed significant negative correlation with circulating adiponectin (p = 0.0195) and a positive correlation with ΔWeight (p = 0.034), ΔBodyFat (0.044) and ΔAdipocyte size (volume, p = 0.012;) in adipocyte-enriched fraction. A whole transcriptome-wide association study (TWAS) on ΔF13A1 vs weight-correlated ΔTranscriptome identified 182 F13A1-associated genes (r > 0.7, p = 0.05) with functions in several biological pathways including cell stress, inflammatory response, activation of cells/leukocytes, angiogenesis and extracellular matrix remodeling. F13A1 did not associate with liver fat accumulation. CONCLUSIONS: F13A1 levels in adipose tissue increase with acquired excess weight and associate with pro-inflammatory, cell stress and tissue remodeling pathways. This supports its role in expansion and inflammation of adipose tissue in obesity.

Assessment of expression and specific activities of transglutaminases TG1, TG2, and FXIII-A during osteoclastogenesis.

Osteoclasts are large multinucleated bone-resorbing cells derived from monocyte/macrophage lineage. Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) drive the multi-stage osteoclastogenesis. Transglutaminases (TGs) are Ca2+- and thiol-dependent acyl transferases and protein crosslinking enzymes. TG enzyme family contains eight catalytically active enzymes TG1-7 and Factor XIII-A (FXIII-A). Recent studies have shown that TG1, TG2, and FXIII-A are present in osteoclasts and that TG2 and FXIII-A regulate osteoclastogenesis. In this study, we examined gene and protein expression and specific activities of TG1, TG2, and FXIII-A during osteoclastogenesis using "Hitomi peptides" in a day-by-day manner. We report that TG activities are highest in the differentiation and early fusion phases and then decrease dramatically. TG activities were upregulated by M-CSF and downregulated by addition of RANKL. FXIII-A was dramatically downregulated by RANKL, suggesting its involvement in M-CSF-mediated precursor commitment phase. TG1 and TG2 proteins were present throughout osteoclastogenesis, suggesting that they may have functions in both differentiation and fusion. In summary, the three TGs likely exert distinct functions at different stages of osteoclastogenesis. Our work also demonstrates that the "Hitomi peptides" are highly specific tools for detection of distinct TGs in a system where multiple TGs are present.

Plasma concentration and cardiovascular effects of intramuscular medetomidine combined with three doses of the peripheral alpha2-antagonist MK-467 in dogs.

OBJECTIVE: We investigated the plasma concentrations and cardiovascular effects of intramuscularly (IM) administered medetomidine, administered alone or with three different doses of MK-467. STUDY DESIGN: Prospective, randomized, open, crossover trial. ANIMALS: Eight purpose-bred healthy Beagle dogs. METHODS: Each dog was administered four treatments: medetomidine 20 µg kg-1 IM alone or mixed in the same syringe with MK-467 (200 µg kg-1, 400 µg kg-1 or 600 µg kg-1). Instrumentation was performed under standardized anaesthesia. The dogs were allowed to recover before measurement of baseline values. Composite sedation scores, cardiovascular variables, i.e., heart rate (HR), cardiac output (CO), mean arterial and central venous blood pressures (MAP and CVP) and arterial blood gases were recorded at baseline and for 60 minutes after treatment. Drug concentrations in venous plasma were analysed. Generalized linear mixed models for repeated measures with post hoc Bonferroni correction were used with statistical significance level set at α=0.05. RESULTS: All treatments initially demonstrated the effects of medetomidine: HR and CO decreased and CVP increased. MAP transiently increased and then significantly decreased from baseline with the two highest MK-467 doses. The cardiovascular effects of medetomidine disappeared more rapidly with MK-467 than with medetomidine alone. With medetomidine alone, sedation scores remained high until the end of the 60 minute follow-up. Maximum concentrations of medetomidine were more rapidly achieved and were higher with MK-467. CONCLUSIONS AND CLINICAL RELEVANCE: Initial haemodynamic effects of medetomidine were not prevented by MK-467, but these effects were attenuated and their duration shortened by MK-467, independently of dose. Absorption of medetomidine was accelerated by MK-467, when administered concomitantly IM, resulting in faster sedation; addition of MK-467 shortened the sedative effect of medetomidine.

Inheritance of antibody specificity V. Anti-2-phenyloxazolone in the mouse.

Antibodies to hapten 2-phenyloxazolone (phOx) of all BALB/c and DBA/2 mice have the same idiotype and the same major (public) isoelectric focusing pattern whose main spectrotype is called Ox-1. Neither of these characteristics could be readily demonstrated in anti-phOx antibodies of C57BL, C3H or LP mice; these antibodies were heterogeneous, and lacked public spectrotypes. Also, a fine specificty difference could be demonstrated between anti-phOx antibodies of BALB/c and C5MBL mice; the latter have a higher relative affinity than the former for a structural analogue of phOx (2-o-iodophenyloxazolone). The three BALB/c characteristics were inherited in congenic and recombinant inbred strains as an allotype-linked block, defining a new VH marker, VHphOx. Murine anti-phOx antibodies were found to exhibit three types of conservatism: (a) Every individual mouse of strains BALB/c, DBA/2 or BAB-14 had an almost indistinguishable IEF pattern. (b) These patterns (and the cross-reactive idiotype) remained virtually unchanged during an immunization course of 70 days. (c) An identical idiotype (and in some cases IEF pattern) was present in mouse strains of five different allogroups.

Post-anesthetic pulmonary edema in two horses.

CASE 1: A two-year old, 462 kg Standard bred horse was anesthetized for arthroscopy and castration. During anesthesia, hyperemia of the mucosal membranes and urticaria were noticed. During 5 hours of anesthesia subcutaneous edema of the eyelids and neck region developed. In the recovery box, the orotracheal (OT) tube was left in situ and secured in place with tape. Following initial attempts to stand, the horse became highly agitated and signs consistent with pulmonary edema developed subsequently. Arterial hypoxemia (PaO(2): 3.7 kPa [28 mmHg]) and hypocapnia (PaCO(2): 3.1 kPa [23 mmHg]) were confirmed. Oxygen and furosemide were administered. The horse was assisted to standing with a sling. Therapy continued with bilateral intra-nasal oxygen insufflation. Ancillary medical therapy included flunixin meglumine, penicillin, gentamycin and dimethylsulfoxide. Following 7 hours of treatment the arterial oxygen tensions began to increase towards normal values. CASE 2: An 11-year old, 528 kg Paint horse was anesthetized for surgery of a submandibular mass. The 4-hour anesthetic period was unremarkable. The OT tube was left in situ for the recovery. During recovery, the horse was slightly agitated and stood after three attempts. Clinical signs consistent with pulmonary edema and arterial hypoxemia (PaO(2): 5 kPa [37.5 mmHg]) subsequently developed following extubation. Respiratory signs resolved with medical therapy, including unilateral nasal oxygen insufflation, furosemide, flunixin meglumine and dimethylsulfoxide. The diagnosis of pulmonary edema in these horses was made by clinical signs and arterial blood-gas analysis. While pulmonary radiographs were not taken to confirm the diagnosis, the clinical signs following anesthesia support the diagnosis in both cases. The etiology of pulmonary edema was most likely multifactorial.

Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin.

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances beta(1)-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.

Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression.

The mechanisms of granulosa cell tumor (GCT) development may involve the dysregulation of signaling pathways downstream of follicle-stimulating hormone, including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this hypothesis, a genetically engineered mouse model was created to derepress the PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K antagonist gene Pten (Pten(flox/flox);Amhr2(cre/+)). The majority of Pten(flox/flox);Amhr2(cre/+) mice featured no ovarian anomalies, but occasionally ( approximately 7%) developed aggressive, anaplastic GCT with pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1 was abrogated in Pten(flox/flox);Amhr2(cre/+) GCT, indicating a mechanism by which GCT cells may increase proliferation and evade apoptosis. To relate these findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT expression were performed on human and equine tumors. Although PTEN loss was not detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has been reported, we tested whether these pathways could synergize in GCT. Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells of a mouse model (Pten(flox/flox);Ctnnb1(flox(ex3)/+);Amhr2(cre/+)) resulted in the development of GCT similar to those observed in Pten(flox/flox);Amhr2(cre/+) mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the ability to spread by seeding into the abdominal cavity. These data indicate a synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the development and progression of GCT and provide the first animal models for metastatic GCT.

Survey of utilization of medetomidine and atipamezole in private veterinary practice in Quebec in 2002.

This survey evaluates early perceptions about the use of medetomidine and atipamezole among veterinary practitioners in Quebec in 2002. Response rate was 23.5%; 71.1% of the practitioners did not use these products because of lack of information (69.3%), unavailability of the drugs in the practice (23.3%), or other reasons (7.3%), including concerns about the safety of alpha-2 agonists. Most veterinarians who used these products (70.4%) used them only rarely. Sedation by medetomidine was qualified as good (44.2%) or excellent (36%), and analgesia as good (46.5%) or average (32.7%). Waking up after atipamezole was qualified as good (47.5%) or excellent (40.9%). These perceptions indicate an opportunity for wider use of the products in veterinary practice. With more education and experience, practitioners could find medetomidine hydrochloride alone or in combination with an opioid useful for sedation, analgesia, and premedication for healthy animals. Reversal with atipamezole hydrochloride is considered effective, when residual sedation is undesirable.

Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability.

BACKGROUND: Patients with an activated renin-angiotensin system (RAS) or genetic alterations of the RAS are at increased risk of myocardial infarction (MI). Administration of ACE inhibitors reduces the risk of MI, and acute coronary syndromes are associated with increased interleukin 6 (IL-6) serum levels. Accordingly, the present study evaluated the expression of angiotensin II (Ang II) in human coronary atherosclerotic plaques and its influence on IL-6 expression in patients with coronary artery disease. METHODS AND RESULTS: Immunohistochemical colocalization of Ang II, ACE, Ang II type 1 (AT(1)) receptor, and IL-6 was examined in coronary arteries from patients with ischemic or dilated cardiomyopathy undergoing heart transplantation (n=12), in atherectomy samples from patients with unstable angina (culprit lesion; n=8), and in ruptured coronary arteries from patients who died of MI (n=13). Synthesis and release of IL-6 was investigated in smooth muscle cells and macrophages after Ang II stimulation. Colocalization of ACE, Ang II, AT(1) receptor, and IL-6 with CD68-positive macrophages was observed at the shoulder region of coronary atherosclerotic plaques and in atherectomy tissue of patients with unstable angina. Ang II was identified in close proximity to the presumed rupture site of human coronary arteries in acute MI. Ang II induced synthesis and release of IL-6 shortly after stimulation in vitro in macrophages and rat smooth muscle cells. CONCLUSIONS: Ang II, AT(1) receptor, and ACE are expressed at strategic sites of human atherosclerotic coronary arteries, suggesting that Ang II is produced primarily by ACE within coronary plaques. The observation that Ang II induces IL-6 and their colocalization with the AT(1) receptor and ACE is consistent with the notion that the RAS may contribute to inflammatory processes within the vascular wall and to the development of acute coronary syndromes.

Mast cell infiltration in acute coronary syndromes: implications for plaque rupture.

OBJECTIVES: To define the role of mast cells in plaque destabilization. BACKGROUND: Inflammation is an essential feature of human coronary plaques. Macrophages and T lymphocytes are considered to contribute to destabilization of the plaques. The role of mast cells in this setting is less well studied. We therefore counted the mast cells in coronary atherectomy specimens from patients with chronic stable angina, unstable angina and severe unstable angina. METHODS: Patients studied had chronic stable angina (group 1, n=11), "stabilized" unstable angina (group 2; Braunwald's class I and II, n=11) and "refractory" unstable angina (group 3; Braunwald's class III, n=7). Samples of culprit lesions (n=29) were obtained by directional atherectomy, snap-frozen and analyzed immunohistochemically. The numbers of mast cells and T lymphocytes per square millimeter squared were counted and the areas containing macrophages and smooth muscle cells were measured by computed planimetry. RESULTS: We found that the numbers of mast cells and T lymphocytes increased from group 1 through groups 2 to 3. Specimens from group 3 also contained the largest numbers of tumor necrosis factor alpha (TNF-alpha)-positive mast cells and of matrix metalloproteinase (MMP)-9 (92 kD gelatinase)-positive macrophages. CONCLUSIONS: Unstable coronary syndromes are associated with increased numbers of mast cells in culprit lesions. Activated mast cells secrete neutral proteases capable of degrading the extracellular matrix and TNF-alpha, capable of stimulating macrophages to synthesize MMP-9. Our observations suggest that mast cells, in addition to macrophages, contribute to matrix degradation and, hence, to progression of coronary syndromes.

Transglutaminase crosslinking of SIBLING proteins in teeth.

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).

The same few V genes account for a majority of oxazolone antibodies in most mouse strains.

The early primary anti-phenyloxazolone antibodies of 12 mouse strains were studied by determining proportions of two defined subsets id495 (the classical phOx idiotype) and id350. Id495-positive antibodies bear an H chain encoded by VHOx1 gene (family Q52) and an L chain usually coded for by VKOx1 but occasionally by other VK genes. Id350-positive antibodies are encoded by a VK gene VK45.1, and usually by a VH gene of the S107 family. All 12 strains (representing nine H-chain and four kappa-chain haplotypes) produced id350-positive anti-phOx antibodies. While id495 is the predominant major subset in the BALB/c response (originally studied), id350 seems to be the predominant subset of early anti-phOx antibodies in the mouse species. The combined proportion of the two subsets varied from ca. 50 to almost 100% of the total in all strains except C57BL.

Combinatorial association of V genes: one VH gene codes for three non-cross-reactive monoclonal antibodies each specific for a different antigen (phoxazolone, NP or gat).

Two anti-phenyloxazolone (phOx3) and one anti-GAT MAbs from C57BL mice are shown to be coded by VH gene 186.2. This gene has been found earlier to code for several anti-NP (NNP) antibodies (Bothwell et al., 1981) and anti-GT antibodies (Rocca-Serra et al., 1983; Carmack and Pincus, 1986). The L chain partner of the VH 186.2 gene is different in anti-NP and anti-GAT antibodies (Bothwell et al., 1981; Rocca-Serra et al., 1983; Carmack and Pincus, 1986); in anti-phOx antibodies two new unrelated kappa chain V regions were found. Both of the new VK genes involved code frequently for anti-phOx antibodies in BALB/c mice but then with different VH genes. We tested five 186.2-coded antibodies for cross-reactions. Four antibodies were specific, one bound only to NNP, one only to phOx and two only to GT (GAT). The fifth antibody (anti-phOx) bound also to NNP, GAT and ABA-HOP though probably with a low affinity. This is the first demonstration that one V gene can code for three different antibody specificities. It emphasizes the role of the combinatorial element in antibody diversity.

Transglutaminase 2--a novel inhibitor of adipogenesis.

Differentiation of preadipocytes to lipid storing adipocytes involves extracellular signaling pathways, matrix remodeling and cytoskeletal changes. A number of factors have been implicated in maintaining the preadipocyte state and preventing their differentiation to adipocytes. We have previously reported that a multifunctional and protein crosslinking enzyme, transglutaminase 2 (TG2) is present in white adipose tissue. In this study, we have investigated TG2 function during adipocyte differentiation. We show that TG2 deficient mouse embryonic fibroblasts (Tgm2-/- MEFs) display increased and accelerated lipid accumulation due to increased expression of major adipogenic transcription factors, PPARγ and C/EBPα. Examination of Pref-1/Dlk1, an early negative regulator of adipogenesis, showed that the Pref-1/Dlk1 protein was completely absent in Tgm2-/- MEFs during early differentiation. Similarly, Tgm2-/- MEFs displayed defective canonical Wnt/ß-catenin signaling with reduced ß-catenin nuclear translocation. TG2 deficiency also resulted in reduced ROCK kinase activity, actin stress fiber formation and increased Akt phosphorylation in MEFs, but did not alter fibronectin matrix levels or solubility. TG2 protein levels were unaltered during adipogenic differentiation, and was found predominantly in the extracellular compartment of MEFs and mouse WAT. Addition of exogenous TG2 to Tgm2+/+ and Tgm2-/- MEFs significantly inhibited lipid accumulation, reduced expression of PPARγ and C/EBPα, promoted the nuclear accumulation of ß-catenin, and recovered Pref-1/Dlk1 protein levels. Our study identifies TG2 as a novel negative regulator of adipogenesis.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

Mast cells accompany microvessels in human coronary atheromas: implications for intimal neovascularization and hemorrhage.

Mast cells have been assigned a role in neovascularization. Therefore, we examined the deep regions of human coronary atheromas, the areas known to be prone to neovascularization, for the presence of mast cells. Specimens of atherosclerotic human coronary intima from 37 autopsy cases with ages of 24-84 years were stained with elastica-van Gieson to detect atheroma formation and with monoclonal antibody against von Willebrand factor to detect neovascularization. Mast cells were detected by staining the atheromas with monoclonal antibodies against the two major proteases of mast cells, tryptase and chymase. Of the 24 coronary atheromas found, 13 contained mast cells in the deep regions. All these 13 deep regions also displayed neovascularization, and the number of microvessels and the number of mast cells around the microvessels correlated strongly with the size of the atheroma. On the other hand, of the 11 deep regions lacking mast cells, only one displayed neovascularization. In the neovascularized areas of the coronary atheromas, the mast cells were in close proximity to the microvessels. All the mast cells contained tryptase, and some of them chymase, both known for their angiogenic and matrix-degrading potential. In light microscopic studies, degranulated mast cells were observed indicating activation of these cells, with release of tryptase and chymase. The selective localization of activated mast cells containing angiogenic factors around newly formed microvessels in human coronary atheromas suggests that mast cells play a role in the neovascularization of these lesions. Moreover, mast cells may also, by virtue of their neutral proteases, injure the microvessels, and thereby produce intraplaque hemorrhages and, ultimately, unstable lesions.

Determination by ellipsometry of the affinity of monoclonal antibodies.

The reaction between monoclonal antibodies and surface-immobilised hapten was studied by ellipsometry, a method allowing absolute measurement of the surface concentration of proteins. Monoclonal antibodies against 2-phenyloxazolone were used and their affinity for the antigen in solution was determined by calculations of the equilibrium constant from data obtained by measuring fluorescence quenching of the hapten due to antibody binding. The binding rate of antibody to surface-immobilised hapten and the dissociation rate of the complex were measured by ellipsometry. The equilibrium constant of the heterogeneous antigen-antibody reaction was determined by a Scatchard plot. The affinity of the antibodies for the antigen was found to be higher in the heterogeneous than in the homogeneous reaction by a factor which varied between different monoclonal antibodies.

Histamine-containing peripheral neuronal and endocrine systems.

An immunohistochemical method was developed to detect histamine in tissues. The aim of this study was to reveal the cellular stores of histamine in the gastrointestinal tract, pituitary, and adrenal gland. Histamine-containing nerve fibers were found in both rat and guinea pig gut. The origin of at least some of these fibers in the rat ileum was the submucous ganglion cell layer. In the rat stomach, numerous enterochromaffin-like cells exhibited histamine immunofluorescence, and endocrine cells in the ileum and jejunum contained histamine. Only mast cells contained histamine in the neurohypophysis. A large number of process-bearing cells in the guinea pig but not in the rat adrenal medulla contained histamine. The study shows that histamine is present in peripheral nerves and endocrine cells in addition to mast cells, and may function as a neurotransmitter or hormone.

Major and minor breakpoint sites of chromosomal translocation t(14;18) in subtypes of non-Hodgkin's lymphomas.

The frequencies of major breakpoint region (MBR) and minor cluster region (MCR) breakpoint sites of t(14;18) were examined by polymerase chain reaction and Southern blotting in 50 non-Hodgkin's lymphomas with cytogenetic evidence of t(14;18). A translocation breakpoint was detected in 41 cases (82%). The MBR was involved in 66%, and the MCR in 16% of the cases. Most cases in the present series were lymphomas with a follicular or diffuse growth pattern, 38 being of the centroblastic/centrocytic type and eight of the centroblastic type. The series also included four lymphomas of probable non-follicular center cell origin. MBR and/or MCR breakpoints were found in all studied lymphoma subtypes and in the majority of these most of the breakpoints were in the MBR and a minor portion of the breakpoints in the MCR. Our results suggest that a breakpoint site is not related to growth pattern or neoplastic cell type in follicular center cell lymphomas with t(14;18).