The presence of p16 CDKN2A protein immunostaining within feline nasal planum squamous cell carcinomas is associated with an increased survival time and the presence of papillomaviral DNA.

In humans, oral SCCs are either caused by papillomavirus (PV) infection or by other carcinogens such as tobacco. As these 2 groups of SCCs have different causes they also have different clinical behaviors. Immunostaining using anti-p16(CDKN2A) protein (p16) antibodies is used to indicate a PV etiology in human oral SCCs and p16-positive SCCs have a more favorable prognosis. The present study investigated whether p16 immunostaining within feline nasal planum SCCs was similarly associated with the presence of PV DNA and with a longer survival time. Intense p16 immunostaining was visible in 32 of 51 (63%) SCCs. In 30 cats with nonexcised SCCs, cats with p16-positive neoplasms had a longer estimated mean survival time (643 days) than cats with p16-negative SCCs (217 days, P = .013). Papillomavirus DNA was amplified more frequently from p16-positive nasal planum SCCs (28 of 32) than p16-negative SCCs (5 of 19, P < .001). The different survival times in cats with p16-positive and p16-negative SCCs suggests that p16 could be a useful prognostic indicator in these common feline cancers. As the clinical behavior of the SCCs can be subdivided using p16 immunostaining, the 2 groups of SCCs may be caused by different factors, supporting a PV etiology in a proportion of feline nasal planum SCCs.

Equine penile squamous cell carcinomas are associated with the presence of equine papillomavirus type 2 DNA sequences.

Forty cases of equine penile disease were screened with polymerase chain reaction for the presence of papillomaviral DNA. Cases consisted of 20 squamous cell carcinomas (average age of horse, 23.9 years) and 20 non-squamous cell carcinoma diseases (average age of horse, 13.3 years). All horses but one originated from the Northeastern United States. Breeds were not recorded. As based on MY09/MY11 consensus primers, DNA sequences from equine papillomavirus type 2 were amplified from 9 of 20 horses (45%) with penile squamous cell carcinoma and only 1 of 20 horses (5%) with non-squamous cell carcinoma penile disease. Equine papillomavirus type 2 DNA was the only papillomaviral DNA amplified from any of the 40 horses. Tissues from the 10 horses in which papillomaviral DNA was detected by polymerase chain reaction were also screened with in situ hybridization and immunohistochemistry. The presence of papillomavirus was demonstrated in a subset of these by in situ hybridization (6 of 10) and immunohistochemistry (1 of 10). This report describes a possible association between equine penile squamous cell carcinomas and equine papillomavirus type 2. This study is also the first report of equine papillomavirus type 2 infection in North American horses.

Intraperitoneal injection of sodium pentobarbital has the potential to elicit pain in adult rats (Rattus norvegicus).

An effective and pain-free killing method is required to achieve the goal of euthanasia, a "good death". Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely accepted technique in laboratory rats, but questions remain regarding pain associated with administration. As PB rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence of pain. The objective of this study was to assess pain associated with IP PB using an appropriate vehicle control. Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 84) were block randomised by sex and strain to receive one of three treatments: 1) 800 mg/kg PB (pH 11), 2) saline or 3) vehicle controls (pH 11 or 12.5). Behavior (Rat Grimace Scale (RGS), writhing, back arching) was evaluated at baseline, before loss of righting reflex (LORR, PB group), and at 80s, 151s and 10 min post-injection (PI; saline and vehicle control groups). In the PB group, mean time to LORR was 78 ± 7.9 seconds. In the vehicle control groups, RGS scores were increased at 151s PI (SD: p = 0.0002, 95%CI 0.73 to 0.20) from baseline, as was relative frequency of writhing (SD: p < 0.0001; Wistar; p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0005, 95%CI 0.71 to 0.18; Wistar: p = 0.0234, 95%CI 0.91 to 0.07) but the relative frequency of writhing did not (p > 0.999). The RGS scores and the relative frequency of writhing remained low in the PB and saline groups (p > 0.05). These results show that, vehicle controls for IP PB result in signs associated with pain, pain may not be experienced following IP PB when LORR occurs quickly, and that the effects of PB limit behavioral pain assessments.

Evidence that extracellular cathepsin D is not responsible for the resorption of cartilage matrix in culture.

Cathepsin D, the major lysosomal aspartic proteinase, is responsible for the autolysis of cartilage at slightly acidic pH, and it has been suspected of making a significant contribution to the breakdown of the living tissue, such as in stimulated by retinol. Our finding, however, has been that neither inhibitory antibodies against cathepsin D, nor chemical inhibition with pepstatin, significantly decreases the rate of degradation of proteoglycan in the organ culture system. Most of the other proteinase inhibitors tested were similarly ineffective, although the EDTA and 1,10-phenanthroline inhibited the resorption by a cytotoxic effect. We conclude that although cartilage matrix degradation has clear characteristics of proteolytic process, the identity of the enzyme(s) responsible remains obscure.

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Structure/function relationships in the inhibition of thimet oligopeptidase by carboxyphenylpropyl-peptides.

Some novel N-[1(RS)-carboxy-3-phenylpropyl]tripeptide p-aminobenzoates have been synthesised as inhibitors of thimet oligopeptidase (EC 3.4.24.15). These compounds are considered to bind as substrate analogues with the Cpp group in S1 and the peptide portion in the S' sites. The most potent inhibitor is Cpp-Ala-Pro-Phe-pAb, which has a Ki = 7 nM. Substitution of Gly for Ala at P1' leads to weaker binding which can be ascribed to increased rotational freedom. Good substrates often have Pro at P2' and Pro is favoured over Ala at this position in the inhibitors, too. When P2' is Pro, Phe is preferred over Tyr and Trp in P3'. The p-aminobenzoate group makes an important contribution to the binding, probably by forming a salt bridge, and removal of the C-terminal negative charge results in much less potent inhibitors.

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.

(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3.

TNF-alpha converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-alpha secretion is involved.

Testing life-history pleiotropy in Caenorhabditis elegans.

Much life-history theory assumes that alleles segregating in natural populations pleiotropically affect life-history traits. This assumption, while plausible, has rarely been tested directly. Here we investigate the genetic relationship between two traits often suggested to be connected by pleiotropy: maternal body size and fertility. We carry out a quantitative trait locus (QTL) analysis on two isolates of the free-living nematode Caenorhabditis elegans, and identify two body size and three fertility QTLs. We find that one of the fertility QTLs colocalizes with the two body size QTLs on Chromosome IV. Further analysis, however, shows that these QTLs are genetically separable. Thus, none of the five body size or fertility QTLs identified here shows detectable pleiotropy for the assayed traits. The evolutionary origin of these QTLs, possible candidate loci, and the significance for life-history evolution are discussed.

Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.

Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI-(GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg(2+)-dependent integrin alpha2beta1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca(2+)-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin alpha2beta1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin alpha2beta1-mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin alpha2beta1 serves to potentiate the signaling effects of GpVI.

Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

Antibodies to a short synthetic peptide cross-react with human recombinant interleukin 1 alpha.

An antiserum to human interleukin 1 alpha has been prepared by immunizing a sheep with a short synthetic peptide (Mr 1427) conjugated to keyhole limpet haemocyanin using the heterobifunctional cross-linking agent N-succinimidyl bromoacetate. The peptide was selected from a highly hydrophilic region corresponding to residues 169-179 of the cDNA-derived sequence. Two additional peptides corresponding to residues 194-207 and 224-233 failed to elicit cross-reacting antibodies.

Erythema multiforme and urticaria. Eruptions induced by chemically related ophthalmic anticholinergic agents.

Erythema multiforme developed in an 80-year-old man following the use of scopolamine hydrobromide ophthalmic drops. The erythema multiforme cleared when the medication was discontinued and recurred on challenge. Later, he was given tropicamide, an anticholinergic ophthalmic preparation that, like scopolamine, has a tropic acid residue. Within 15 minutes an immediate hypersensitivity reaction with generalized urticaria developed in the patient.

The retention and distribution in the rabbit knee of a radionuclide complexed with a lipophilic chelator in liposomes.

The application of radiosynovectomy to patients with rheumatoid arthritis has been severely restricted by the difficulty of preventing leakage of the radioisotope from the joint cavity. We have synthesised a lipophilic chelator, 3-cholesteryl 6-[N'-iminobis(ethylenenitrilo)-tetraacetic acid]hexyl ether (Chol-DTTA) which can complex with a variety of beta-emitting radionuclides and is incorporated into the lipid phase of liposomes. The retention in the synovial cavities of rabbit knees of liposomes containing Chol-DTTA, complexed with the gamma-emitting tracer 51Cr, has been measured over a period of 21 days and compared with colloidal and water-soluble preparations. The distribution of the radionuclide between the tissues of the joint was also examined. Results show retention of 51Cr delivered in chelator liposomes to be greater than 99% after 24 h. At this time, over 93% of the radioactivity had become associated with the synovium. We conclude that chelator liposomes offer considerable promise as vehicles for radioisotopes in radiosynovectomy.

Effect of the intra-articular injection of lutetium-177 in chelator liposomes on the progress of an experimental arthritis in rabbits.

The treatment of rheumatoid arthritis by radiosynovectomy has been restricted by the difficulty of preventing leakage of the radioisotope from the joint cavity. We have previously shown that this leakage can be reduced to very low levels by delivering the radioisotope in liposomes containing the lipophilic chelator, 3-cholesteryl 6-[N'-iminobis-(ethylenenitrilo)tetraacetic acid]hexyl ether. The present study investigates the effectiveness of the beta-emitting isotope lutetium-177, delivered in chelator liposomes, in treating an experimental arthritis in rabbits. Chelator liposomes containing 0.35 mCi, 0.175 mCi Or 0.087 mCi of the isotope were injected into the synovial cavities of the knees of rabbits with an established experimental arthritis. The retention of the lutetium and the progress of the arthritis were followed for 47 days, and samples of the joint tissues were taken for histology at the end of the experiment. Results showed that losses of radioactivity averaged less than 1% per day over 47 days and that joints treated with 0.175 mCi showed significant reductions in both diameter and surface temperature compared with controls treated with a non-radioactive preparation. Post-mortem histology revealed that, whereas control joints showed a highly active synovitis, synovia of joints treated with 0.175 or 0.35 mCi lutetium-177 had very little inflammatory activity. Although some joints which had received 0.35 mCi showed signs of damage to the articular cartilage, this damage was not apparent wih either of the two lower doses. We conclude that, in this animal model, chelator liposomes complexed with a suitable radioisotope are capable of effecting an efficient synovectomy.

The effect of side chain structure on the biochemical and therapeutic properties of intra-articular dexamethasone 21-esters.

The prolonged anti-rheumatic effects produced by some higher 21-esters of intra-articular corticosteroids have been ascribed to their low aqueous solubility or, alternatively, to their slow release of free (21-OH) steroid in the inflamed synovium. Experiments were designed to test this hypothesis. Twelve 21-carboxyl esters of dexamethasone and [3H]dexamethasone were prepared. Their side-chain structures were chosen to provide systematic steric hindrance of the scissile bond. Four dexamethasone/[3H]dexamethasone 21-carbamates were also prepared. When incubated with a 10% (w/v) homogenate of rabbit synovial tissue, esters providing steric hindrance, e.g. t-butylacetate, were more slowly hydrolysed than those which were linear, e.g. n-hexanoate, or cyclic e.g. cyclohexane acetate. Carbamate esters remained unhydrolysed during 24 hours' incubation. The partition coefficients of these compounds, derived using reversed-phase thin-layer chromatography and hydrophobic fragmental constants, were not correlated with their hydrolysis rates. Isomeric 21-substituents had similar partition coefficients. The affinity of the isomers, dexamethasone n-hexanoate and dexamethasone t-butylacetate, for the glucocorticoid receptor of mouse fibroblast cytosol, was determined by a competitive binding assay using [3H]triamcinolone acetonide. Dexamethasone t-butylacetate had 1/10 binding affinity relative to that of dexamethasone. Dexamethasone n-hexanoate was inactive. The therapeutic activities of dexamethasone n-hexanoate and dexamethasone t-butylacetate were compared at a single dose (2 mg), injected into experimentally-arthritic rabbit knee joints. These preparations reduced the swelling and histopathological changes in the treated joints by the same extent, indicating that the local anti-rheumatic activity of corticosteroid 21-esters is unrelated to their hydrolysis rates in vitro.

The hydrolysis of cortisol 21-esters by a homogenate of inflamed rabbit synovium and by rheumatoid synovial fluid.

Long chain esters of cortisol have shown prolonged anti-inflammatory activity in both clinical and animal studies. This effect has been ascribed to the decreased water-solubility of the higher esters, but an alternative explanation is that the higher esters are hydrolysed more slowly to free steroid by the synovial tissue enzymes. In order to investigate the influence of chain length on hydrolysis rate we synthesized a series of cortisol 21-esters. When incubated in a 0.1% (w/v) homogenate of inflamed rabbit synovial tissue the esters with chain lengths of 4, 6, 8 and 10 carbon atoms were hydrolysed much faster than those with 2, 12, 14 and 16 carbon atoms. At tissue concentrations of 10% (w/v), however, the breakdown of cortisol acetate was greatly accelerated, whereas cortisol palmitate remained quite stable. Although cortisol esters were hydrolysed in 50% (v/v) rheumatoid synovial fluid, the rates of hydrolysis were relatively slow. The chain length dependence was similar to that seen with the tissue homogenate.

Robot-assisted minimally invasive Kasai portoenterostomy: a survival porcine study.

BACKGROUND: Major enhancements offered by robotic surgery for minimally invasive procedure include tremor filtration, motion scaling, and the addition of a wrist to the instrument. Minor enhancements include indexing as well as safe and rapid instrument exchange. A benefit associated with any endoscopic procedure is magnification. It was hypothesized that these enhancements would allow the performance of complex gastrointestinal surgery. METHODS: Eight survival pigs (weight, 2.5-8 kg) underwent a robotically assisted minimally invasive portoenterostomy. The procedure was analogous to the Kasai portoenterostomy for biliary atresia usually performed for human patients at the age of 4 to 12 weeks. RESULTS: Five of the eight animals survived for more than 1 month after the operation, returning to normal eating and bowel habits in 2 to 3 days. None were jaundiced. All laboratory values were normal. At 1 month, the animals were killed. There was no anastomotic stenosis at either the end-to-side enteroenterostomy or the portoenterostomy. Histologically, the anastomoses were well healed. CONCLUSION: Computer-assisted robot-enhanced technology allows complex gastrointestinal surgery to be performed using minimally invasive techniques.

The use of a chelating derivative of alpha melanocyte stimulating hormone for the clinical imaging of malignant melanoma.

A novel chelating derivative of alpha melanocyte stimulating hormone, bis MSH-DTPA, has been used for the diagnostic targeting of malignant melanoma. 15 patients were investigated of whom nine were shown by other means to have active disease at the time of the scan. Tumours were imaged in all of these nine patients. Of a total of 46 lesions over 10 mm encountered, 41 (89%) were imaged. There were no false positives and in two cases bisMSH-DTPA was instrumental in reversing diagnoses made using ultrasound. Derivatives of melanocyte stimulating hormone may be of considerable value in targeting melanomas.