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Introduction: Cytokine growth factors have been ineffective in infected wounds (>105 bacteria/g of tissue) for several reasons. Bacteria degrade the polypeptides themselves, and this effect is accentuated in the presence of tissue cells when excessive matrix metalloproteinases are produced. Also, the growth factor receptors for several cytokines have been shown to have decreased expression in chronic wounds.Materials and Methods: The effects of genomics-based KGF-2 (repifermin) on healing of infected wounds were examined in 3 experiments using a rodent model of a chronically infected (>105 bacteria/g of tissue) granulating wound. Serial wound area measurements were compared among animals treated with vehicle, 2 doses of KGF-2, and KGF-2 in combination with GM-CSF. Binding of biotinylated KGF-2 was compared with binding of a biotinylated irrelevant protein control in sections of both noninfected and infected granulation tissue. Serial biopsies of the wounds for quantitative bacteriology confirmed the wounds were infected throughout the experiments.Results: In all 3 experiments, infected wounds healed faster when topically treated with KGF-2 (P <0.05). The effect was seen most dramatically toward the end of the wound closure curve when the wounds were 75% to 90% closed. Using GM-CSF in combination or in sequence with KGF-2 did not enhance the activity of KGF-2 alone. There was no clear decrease in KGF-2 binding in infected versus noninfected wounds. In contrast, sections incubated with the control-irrelevant protein were uniformly negative. Bacterial counts confirmed the wounds remained at a bacterial tissue level of >105 organisms/g.Conclusion: Repifermin (genomics-based KGF-2) appears more robust than other reported growth factors in the presence of high levels of bacteria. This appears to be the result of its lesser degree of degradation in the presence of bacteria and tissue fibroblasts and the ability of its receptors to remain functional in infected tissue. [ABSTRACT FROM AUTHOR]

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes 1 . Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.

Despite emerging evidence for the comorbidity of posttraumatic stress disorder (PTSD) and migraine, few studies have examined the relation of PTSD and migraine, particularly among clinical populations at-risk for both conditions (e.g., substance-dependent patients). This study examined the role of PTSD symptoms in migraine and headache-related disability within a sample of 153 substance-dependent inpatients (37.25% female, Mean age 36.46). PTSD symptoms predicted both migraine and headache-related disability above and beyond gender, depression and anxiety symptoms, the experience of a Criterion A traumatic event, and current alcohol use disorder. Findings highlight the strong association between migraine and PTSD symptoms in a unique population at risk for both conditions.

Objective: To determine the proportion of dog breeders who fed diets meeting the Association of American Feed Control Officials regulations for nutritional adequacy for reproduction and growth and to investigate factors that influenced feeding practices of breeders., Design: Web-based cross-sectional survey., Sample: 2,067 dog breeders from the United States and Canada., Procedures: A self-administered, anonymous, Web-based questionnaire was used to collect information on breeder demographics and feeding practices during 3 life stages of dogs: adult maintenance for nonpregnant dogs, gestation-lactation, and puppy growth. Appropriateness of commercial diets for each life stage was determined by respondent-reported nutritional adequacy statements on product labels. Data were also collected regarding breeder criteria for diet selection and sources of nutrition information., Results: A substantial number of breeders reported feeding commercial diets not intended for that life stage during gestation-lactation (126/746 [16.9%]) and puppy growth (57/652 [8.7%]). Additionally, approximately one-seventh of breeders reported feeding home-prepared diets for ≥ 1 life stage. Unsubstantiated health and marketing information influenced diet selection of many breeders. Veterinarians, although generally viewed as a trusted source of nutrition information, were consulted by only 823 of 1,669 (49.3%) breeders and were viewed less favorably by breeders feeding home-prepared diets, compared with the opinion of breeders feeding commercial diets., Conclusions and Clinical Relevance: Veterinarians should consider taking a more proactive role in directing dog breeders and other pet owners toward scientifically substantiated sources of diet information and in explaining the importance of current nutritional standards for reproduction and early development of dogs.

Surface proteins in Gram-positive bacteria are anchored to the cell wall by the action of sortase enzymes. The Staphylococcus aureus sortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG. To understand how SrtA binds this sequence, we carried out NMR studies of new peptidyl-cyanoalkene and peptidyl-sulfhydryl inhibitors that contain the sorting signal sequence LPAT. These studies combined with amino acid mutagenesis identified a catalytically important and conserved binding surface formed by residues A118, T180, and I182. Compatible with its recently proposed role as a general base, R197 is also shown to be required for catalysis.

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.

Inhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production in vitro in human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-alpha production. Using compounds of two distinct chemical structural classes, a quinazolinedione (CP-77059) and a 4 arylpyrrolidinone (rolipram), we show here that PDE-IV-specific inhibitors are also potent in suppressing LPS-induced TNF-alpha production in vitro in sodium periodate-elicited murine macrophages (IC50s of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-alpha induced by a sublethal LPS injection; (ii) LPS-induced endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublethal (5 micrograms/mouse) injection of LPS, serum TNF-alpha levels in mice peaked sharply, reaching concentrations of 3-12 ng/ml 90 min after injection. In this sublethal LPS assay, CP-77059 was about 30 times more potent than rolipram, with a minimum effective dose of 0.1 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro IC50s for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77059 at relatively high doses of 30 and 10 mg/kg, respectively, significantly reduced serum TNF-alpha levels, and also inhibited mortality 66%. In the LPS/galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by co-injection with galactosamine, only 0.1 microgram of LPS/mouse is necessary for serum TNF-alpha elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-alpha and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-alpha response (without a serum TNF-alpha elevation), rolipram significantly inhibited paw swelling as well as localized TNF-alpha levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-alpha elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-alpha production and inflammatory responses in murine systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

Proliferating cell nuclear antigen (PCNA) was evaluated as a marker of cell proliferation in formalin-fixed rat liver tissue through a comparative study with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). The comparison was conducted through the introduction of a dual immunohistochemical procedure that allows the simultaneous detection of the two antigens. The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous. The method presented here for the simultaneous labeling of PCNA and BrdU antigens may have utility in studies of cell cycle perturbations.

5-Fluorouracil (5-FUra), 5-deoxy-5-fluorouridine (5'dFUrd), and 5-fluorouridine were compared for their relative antitumor activity, their capacity to inhibit leukocyte exudation and macrophage (macrophage) killing of tumor cells in vivo and in vitro, and their ability to induce leukopenia and monocytopenia. 5'dFUrd was less toxic than 5-FUra and exhibited anti-Ehrlich ascites activity over a wider range of drug doses. Inflammatory exudates induced by thioglycollate or pyran were inhibited up to 91% by prior 5-FUra injection but were inhibited not more than 62% by 5'dFUrd. Pyran-induced macrophage inhibition of Ehrlich ascites proliferation in vivo was diminished up to 5-fold by 5-FUra but was never diminished more than 2-fold by 5'dFUrd, while neither agent suppressed in vitro macrophage cytotoxicity of in vivo pyran-activated macrophage. At high doses, 5-FUra reduced white blood cell counts 73%, in contrast to the 8% reduction caused by 5'dFUrd, while at their optimal anti-Ehrlich ascites doses, 5-FUra and 5'dFUrd both lowered white blood cell counts by only 20%. However, 5-FUra caused a severe monocytopenia not seen in animals given injections of comparable doses of 5'dFUrd. Therefore, 5-FUra appeared to inhibit the inflammatory response and antitumor activity by inhibiting the influx of immature macrophage into the peritoneal cavity, not by inhibiting the function of mature effector cells.

Normal BALB/c mice were assessed for 2,4-dinitrofluorobenzene (DNFB)-induced contact sensitivity following adoptive transfer of macrophages (Mo). T cells, or their derived products, from normal or tumor-bearing hosts (TBH). Contact sensitivity (CS) was measured by a quantitative radioisotopic ear assay, a total in vivo system based on localization of IP-injected iodinated human serum albumin [( 125I]HSA) in the DNFB-challenged ear. Adoptive transfer of low or high doses of TBH T cells or their derived supernatants into normal recipients suppressed their responsiveness, while Mo supernatants enhanced it. Moreover, in all cases adoptive transfer of TBH cells or supernatants resulted in a lower CS response than did their normal counterparts. These results further corroborate our previous in vitro data indicating that T cells, or Mo and T cell soluble products, possess immunoregulatory capabilities in vivo.