PACAP-PAC1R modulates fear extinction via the ventromedial hypothalamus.

Exposure to traumatic stress can lead to fear dysregulation, which has been associated with posttraumatic stress disorder (PTSD). Previous work showed that a polymorphism in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide) system is associated with PTSD risk in women, and PACAP (ADCYAP1)-PAC1R (ADCYAP1R1) are highly expressed in the hypothalamus. Here, we show that female mice subjected to acute stress immobilization (IMO) have fear extinction impairments related to Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP-c-Fos downregulation in the Medial Amygdala (MeA), and PACAP-FosB/ΔFosB upregulation in the Ventromedial Hypothalamus dorsomedial part (VMHdm). DREADD-mediated inhibition of MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 polymorphism have impaired fear extinction.

Cytotoxicity of electro-surgical smoke produced in an anoxic environment.

BACKGROUND: The effect on cell viability of smoke produced during high-frequency electro-surgery has not been previously reported. The aim of this study was to produce smoke in vitro, in a closed environment similar to that encountered in minimal access surgery, and to test its cytotoxic effects on cultured cells. METHODS: Pig liver was cut repeatedly with an electro-surgical hook knife, and the smoke generated was collected and equilibrated with cell culture medium. MCF-7 human breast carcinoma cells were exposed briefly to various dilutions of this medium and tested for clonogenicity. RESULTS: Electro-surgical smoke produced in a helium environment reduced the clonogenicity of the MCF-7 human breast carcinoma cells in a dose-dependent manner, falling to 30% when the cells were exposed to undiluted medium for 15 minutes. CONCLUSIONS: We conclude that electro-surgical smoke is cytotoxic. The sublethal effects at lower dilutions are currently being investigated.

Time-dependent photodynamic damage to blood vessels: correlation with serum photosensitizer levels.

We have used the technique of dynamic capillaroscopy to study the time-course of photodynamic vascular occlusion in mice injected intraperitoneally with either of two photosensitizers; hematoporphyrin esters (HPE) or meso-tetrahydroxyphenyl chlorin (mTHPC). The peak of vascular occlusion induced by HPE coincided in time with peak serum levels of this photosensitizer (about 3 h after injection). However, there was also a second peak of occlusive activity at about 12 h after injection, at which time serum HPE was falling monotonically. In the case of mTHPC, no peak of occlusive activity was seen at 3 h after injection, even though the serum levels of this photosensitizer, like those of HPE, were highest around this time. Instead, a steady rise in photosensitizing activity was observed, peaking at 11 h. This decoupling between serum drug levels and vascular photosensitization--partial for HPE and complete for mTHPC-suggests that direct photosensitization of endothelial cells is unlikely wholly to explain the vascular collapse. Instead, there must be either another compartment that accumulates photosensitizer more slowly and in which photodynamic activity has an indirect effect on the blood capillaries or a slow metabolic transformation of mTHPC into a more active sensitizer.

Photosensitizer targeting in photodynamic therapy. II. Conjugates of haematoporphyrin with serum lipoproteins.

Conjugates between haematoporphyrin (HP) and human low-density lipoprotein (LDL), human high-density lipoprotein (HDL) and bovine HDL have been prepared, purified and characterized. HP-LDL is aggregated possibly via interparticle apoB protein cross-linking. HP-HDL human and bovine conjugates show different degrees of intraparticle apoA polypeptide cross-linking. Receptor-mediated endocytosis of HP-LDL by NIH 3T3 cells is inferred from the increased uptake observed when LDL receptors are upregulated. HP-LDL uptake into HT29 cells faces competition from unlabelled LDL, albeit at rather high doses. HP-HDL uptake is also inhibited by LDL, suggesting that both lipoprotein conjugates may have cell-surface binding sites in addition to the specific LDL (apoB) receptor. J774.2 macrophages avidly accumulate HP-LDL, retaining most of the fluorescence and some of the protein while degrading the remainder. Oxidized LDL species compete in these processes, with the major effect on protein degradation. Chloroquine has little effect on the fluorescence uptake but inhibits protein degradation (and hence enhances protein accumulation). HP-HDL is also avidly taken up by J774.2 cells, but in the case of the bovine material with a sigmoidal concentration dependence. This is consistent with prior aggregation before the particles can be endocytosed. P388.D1 cells, which appear to be less activated than the J774.2 line, take up less fluorescence and retain and degrade less protein, but still to higher extents than observed for non-phagocytic cells. We conclude that photosensitizer-lipoprotein conjugates can be taken up in large amounts by cells possessing scavenger receptors and/or phagocytic activity, and that this may be a means of targeting photodynamic therapy.

Photosensitizer targeting in photodynamic therapy. I. Conjugates of haematoporphyrin with albumin and transferrin.

Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly-L-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer-protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.

The prognostic significance of the accumulation of p53 tumour-suppressor gene protein in gastric adenocarcinoma.

We have studied the expression of p53 in 206 patients with gastric adenocarcinomas. A standard immunohistochemical technique employing the CM-1 anti-p53 polyclonal antibody was applied to the routinely fixed and paraffin-embedded material from these tumours; overexpression of p53 was defined as positive nuclear staining: 46% (94/206) of gastric carcinomas expressed high levels of p53. There was no significant correlation between p53 positivity and the tumour grade, growth pattern, the Lauren type or lymph node metastases. Correlation with disease stage was only marginally significant (P = 0.05). Life table analysis revealed a highly significant association between p53 expression and survival (P = 0.0062), the odds ratio of death being 1.89 (95% confidence interval 1.33-2.69). The overall 5-year survival of patients with p53-positive tumours was 3% compared with 16% for those with p53-negative tumours (median survival time being 5.6 and 11.4 months respectively). These data suggest that overexpression of the p53 oncoprotein is an independent marker of shortened survival in gastric cancer patients.

On the mechanism of the tumour-localising effect in photodynamic therapy.

The proposed mechanisms by which tumours concentrate photosensitisers are reviewed. Tumour-associated macrophages have been shown by others to accumulate up to nine times the level of porphyrins as do tumour cells. Macrophages also take up and degrade oxidised or otherwise modified low-density lipoprotein (LDL). We propose that the interaction of photosensitisers with LDL is an important factor, leading to accumulation in macrophages. Uptake into these cells via liposomes and high-density lipoprotein is also possible. There may be three separate mechanisms for tumour destruction in photodynamic therapy: (i) direct damage to tumour cells; (ii) damage to the endothelial cells of the tumour microvasculature; and (iii) macrophage-mediated immune infiltration of the tumour. The association of photosensitisers with lipoproteins may accentuate the latter two (endothelial cells can also accumulate modified lipoproteins). Accumulation in macrophages may also largely explain the high porphyrin retention observed in atheromatous plaques.

Immunoscintigraphy of primary colorectal cancers with indium-111 monoclonal antibody B72.3.

Immunoscintigraphy with CYT-103, an 111indium-labelled immunoconjugate of B72.3, was evaluated in 10 patients before surgery for suspected or biopsy-proven primary colorectal cancer. The imaging results were compared with computed tomography (CT) findings at surgery, histopathology and immunohistochemistry. There were no adverse reactions following the administration of 1.0 mg 111In-CYT-103. Surgical and pathological findings identified 15 sites of disease (10 primary and five metastatic) and all but one lesion (severe dysplasia) were malignant. CT detected nine of 14 sites of malignancy compared to 12 as identified by immunoscintigraphy. It failed to detect two primary lesions and one case of peritoneal metastasis, all of which were imaged by CYT-103. Both imaging modalities failed to detect two of three cases with lymph node metastases and the dysplastic lesion (true negatives). The results indicate that 111In-CYT-103 imaging exhibits high sensitivity and specificity in the detection of primary and secondary lesions in patients with colorectal cancer.

Dynamic capillaroscopy: a minimally invasive technique for assessing photodynamic effects in vivo.

A noninvasive method for visualizing the microvasculature in the mouse tail is described, consisting of a custom-built microscope with through-lens illumination. The microscope is fitted with a television camera and images can be recorded on videotape and displayed on a television monitor. Blood vessels are imaged as columns of red blood cells, in which flow is clearly observed. Administration of photosensitizers and illumination with the standard light source produces no observable photodynamic effect on blood flow. The combination of photosensitizer and a more intense light source (either broadband light from a filtered mercury arc or red light from a laser) causes photodynamic cessation of flow within a few minutes. The magnitude of the effect is dependent on the dose and nature of the photosensitizer, the delay after photosensitization and the match between the laser light and the absorption spectra of the photosensitizers in the red region. We conclude that the technique yields results consistent with the known photodynamic effects of the photosensitizers in tumors and propose its use as an initial screening method in vivo, as a means of conducting pharmacokinetic experiments and as an assay of prolonged cutaneous photosensitivity.

Enzyme-linked immunosorbent assay for p53 in gastrointestinal malignancy: comparison with immunohistochemistry.

Mutations in the p53 nuclear oncogene occur frequently in a wide spectrum of human malignancies and the mutant protein may prove to be a useful diagnostic or prognostic marker. It can be detected in fixed tissues by immunohistochemistry, but the type of fixative and conditions of fixation used can introduce variability. For routine clinical use, a method of analysis which is more easily standardized would, therefore, be of benefit. A two-site enzyme-linked immunosorbent assay (ELISA) was used to measure the level of p53 protein in soluble extracts from 20 gastrointestinal cancers (11 colonic, nine gastric). Immunohistochemistry was also performed on the paraffin-embedded sections of these tumours and the results of the two assays were compared. ELISA detected p53 at various levels in 10 cases, all of which were also positive by immunohistochemistry. Of the other 10, eight were immunohistochemically negative but two were positive. When the immunohistochemically positive specimens were ranked by scoring the degree of staining, there was a highly significant correlation with the quantitative ELISA results. Our study shows that the ELISA is sensitive and highly specific. It offers an alternative and simple method of assessing the p53 status in human tissues.

Monoclonal antibodies to cytoskeletal proteins: an immunohistochemical investigation of human colon cancer.

Monoclonal antibodies raised to a number of microfilament-associated proteins were shown to recognize the appropriate proteins in extracts from human colon tissue. They were then used in an immunohistochemical study of normal colonic mucosa, adenomas, and adenocarcinomas. A strong reaction was seen in stromal cells within the tumours (both adenomas and adenocarcinomas) when frozen sections were stained with antibodies to filamin and caldesmon. In addition, a similar reaction was seen in the adenocarcinomas when stained with antibodies to talin and gelsolin. We believe that immunohistochemical staining with these antibodies reveals a tumour-induced process in the surrounding cells, possibly related to a host response to tumours.

Expression of p53 protein in normal, dysplastic, and malignant gastric mucosa: an immunohistochemical study.

Mutations in the p53 nuclear oncogene are the most frequent genetic abnormalities encountered in human malignancies. Using the polyclonal antibody CM-1, we have examined the expression of the p53 oncoprotein immunohistochemically in archival material of normal, dysplastic, and malignant gastric mucosa. Abnormal expression of this protein was not observed in biopsies of normal gastric tissue (n = 30) but was detected in 22 of the 36 gastric cancers analysed (61 per cent). Nuclear staining was diffuse in 15 of the positive cancer cases, the remaining seven showing a more varied heterogeneous staining pattern. Abnormal p53 protein was not detected in mild (n = 14) or moderate (n = 16) gastric dysplasia but was present in 3 out of 15 severe dysplasia cases. The results suggest that expression of the p53 oncoprotein is a common finding in gastric cancer and occurs as a late event in the malignant transformation process.

Tamoxifen metabolism is altered by simultaneous administration of medroxyprogesterone acetate in breast cancer patients.

We have measured the levels of tamoxifen and three of its metabolites in the blood of patients receiving tamoxifen alone or combination therapy with tamoxifen and medroxyprogesterone acetate. Our results indicate that addition of the progestogen significantly alters the metabolism of tamoxifen over a six-month period. We suggest that the interaction between these drugs may involve additional sites (probably hepatic) besides the desired target tumour.

Semi-permeable microcapsules for cell culture: ultra-structural characterization.

Microcapsules made from alginate-poly(L-lysine)-alginate membranes have been studied as vehicles for cell culture in a number of laboratories. We have examined their permeability, robustness and ultrastructure in detail. Permeability to globular proteins could be controlled by using poly-lysine of different mean MW in their construction. However, this parameter also affected the degree to which microencapsulated living cells leaked out of the capsules during and after preparation. Poly-lysine of low MW produced a relatively permeable and robust membrane whereas a high MW produced capsules with the reverse characteristics. A MW of 22,000 appears to be optimal in forming robust capsules which are relatively impermeable to high MW species such as immunoglobulins. The structure of the semipermeable membrane was investigated by electron microscopy and found to be complex but entirely consistent with the data on protein permeability and cell leakage. Microcapsules were not disrupted by gentle treatment with trypsin or chelating agents but dissolved with the addition of heparin, sodium dodecyl sulphate or sodium hydroxide. Empty microcapsules implanted into the peritoneal cavity of rats elicited a host cellular reaction but remained intact for at least three months.

Microencapsulation of human cells: its effects on growth of normal and tumour cells in vitro.

The growth kinetics of established human colorectal tumour cell lines (HT29, HT115 and COLO 320DM) and human diploid fibroblasts (Flow 2002) were studied in conventional culture and in microcapsules formed from alginate-poly(L-lysine)-alginate membranes. The tumour lines grew rapidly in microcapsules but, in the case of the substrate-adherent lines HT29 and HT115, only after a prolonged lag phase. This phase was reduced by serial passage in microcapsules. The anchorage-independent line COLO 320DM showed no lengthening in lag phase. Microencapsulated fibroblasts underwent negligible growth but remained viable. Some evidence for functional differentiation (microvilli, cell-cell junctions) of the tumour line HT115 within the microcapsules was observed. We conclude that the use of microcapsules provides an alternative system with some advantages for the study of human cancer and its metastases in vitro.

Bradykinin blocks the action of EGF, but not PDGF, on fibroblast division.

Quiescent fibroblasts derived from human fetal lung can be stimulated to reinitiate DNA synthesis by sequential addition of 3 nM IGF-1 and a low concentration (8 pM) of EGF or by continuous exposure to 10% fetal calf serum or 10 ng/ml PDGF. Bradykinin blocks the IGF-1 and EGF-dependent signals without affecting the response to serum or PDGF. It activates protein kinase C and its anti-mitogenic effect is abolished after this kinase has been down-regulated. Bradykinin has no effect on the binding affinity of the EGF receptor whereas phorbol ester induces its 'transmodulation' to low affinity.

Mu-receptor specificity of the opioid peptide irreversible reagent, [3H]DALECK.

A novel affinity reagent DALECK, i.e. D-Ala2-Leu5-enkephalin with a C-terminal chloromethyl ketone group, was previously synthesized in normal and in tritiated form and shown to react irreversibly at opioid receptors, with some evidence for selectivity for the mu subtype. DALECK tritiated in its phenolic group has been synthesized at 13-fold higher specific radioactivity than in the previous study. In the irreversible reaction of this product at pH 8.1 with rat brain membranes it was confirmed that only one polypeptide there is labelled, of apparent Mr 58,000. Competition between this reaction and ligands highly selective for the mu, delta or kappa binding sites yielded curves demonstrating the very high selectivity of the DALECK irreversible reaction for the mu site. The results provide evidence that the mu opioid receptor protein contains only one type of binding subunit, whose apparent Mr is 58,000, this size being dependent upon the conditions used in the gel electrophoresis and being higher when stringent conditions which would reduce all internal disulphide bonds are applied.

Differential recovery of antibody production potential after sublethal, whole-body irradiation of mice.

Mice were given single injections of sheep erythrocytes (SE) or polyvinylpyrrolidone (PVP) at various times after sublethal, whole-body irradiation (550 rad 60CO) and direct, antigen-specific, plaque-forming cell (PFC) responses were quantified. Irradiated mice did not respond to SE or PVP when immunized 15 d postirradiation (PI); by day 30 PI, the responses by irradiated mice were 40-126% of normal to SE and 3-38% of normal to PVP. The impaired recovery after irradiation of immune responses to PVP was not due to altered antigen dose requirements or altered time of peak PFC response and occurred after irradiation of mice by doses as low as 200 rad. Both athymic and euthymic mice had impaired responses to PVP after whole-body irradiation. The impaired response of irradiated mice to PVP was repaired by adoptive transfer of normal bone marrow, fetal liver, or spleen cells and also by spleen cell preparations enriched in Ig+ cells but not by spleen cell preparations enriched in Thy.1+ or Ig- cells. With the aid of additional antigens it was observed that by day 30 PI, mice had recovered ability to respond to the T-cell-dependent antigen SE and the T-cell-independent type-1 antigens 2,4,6-trinitrophenyl-Brucella abortus and butanol-extracted bacterial lipopolysaccharide, but at that time they gave impaired responses to the T-cell-independent type-2 antigens PVP, type III pneumococcal polysaccharide, and phenol-extracted bacterial lipopolysaccharide; they had an immune response pattern similar to that of CBA/N mice having an X-linked immunodeficiency.

Identification of an opioid receptor subunit carrying the mu binding site.

The enkephalin affinity reagent [3H]Tyr-D-Ala-Gly-Phe-Leu-CH2Cl [( 3H]DALECK) was synthesized. It exhibited high-affinity reversible binding, at pH 7.4, to both mu and delta opioid receptor sites in rat brain membranes. At pH 8.1, nanomolar levels of [3H]DALECK produced an irreversible labeling in synaptic membranes, essentially only in one subunit of 58 000 daltons. The irreversible phase of the reaction reduced the subsequent binding of a mu-selective enkephalin derivative but not that of a delta-selective one. It is concluded that a mu subunit of the opioid receptor exists, can be alkylated specifically, and is of Mr 58 000.