Microbiology of equine wounds and evidence of bacterial biofilms.

Horse wounds have a high risk of becoming infected due to their environment. Infected wounds harbour diverse populations of microorganisms, however in some cases these microorganisms can be difficult to identify and fail to respond to antibiotic treatment, resulting in chronic non-healing wounds. In human wounds this has been attributed to the ability of bacteria to survive in a biofilm phenotypic state. Biofilms are known to delay wound healing, principally due to their recalcitrance towards antimicrobial therapies and components of the innate immune response. This study describes the presence of bacterial biofilms within equine wounds. Thirteen 8-mm diameter tissue samples were collected from (n=18) chronic wounds. Following histological staining, samples were observed for evidence of biofilms. Fifty one wounds and control skin sites were sampled using sterile swabs. Control skin sites were on the uninjured side of the horse at the same anatomical location as the wound. The isolated bacteria were cultured aerobically and anaerobically. The biofilm forming potential of all the isolated bacteria was determined using a standard crystal violet microtitre plate assay. Stained tissue samples provided evidence of biofilms within 61.5% (8 out of 13) equine wounds. In total 340 bacterial isolates were identified from all the equine wound and skin samples. Pseudomonas aeruginosa and Enterococcus faecium were the most predominantly isolated bacterial species from equine wound and skin samples respectively. Staphylococcus was the most commonly isolated genus in both environments. Bacteria cultured from chronic and acute wounds showed significantly (P<0.05) higher biofilm forming potential than bacteria isolated from skin. This paper highlights preliminary evidence supporting the presence of biofilms and a high microbial diversity in equine chronic wounds. The presence of biofilms in equine wounds partly explains the reluctance of many lower limb wounds to heal. Non-healing limb wounds in horses are a well documented welfare and economic concern. This knowledge can be used to shape future treatments in order to increase the healing rate and decrease the costs and suffering associate with equine wounds.

Distal margin requirements after preoperative chemoradiotherapy for distal rectal carcinomas: are < or = 1 cm distal margins sufficient?

BACKGROUND: Sphincter-sparing alternatives to abdominoperineal resection (APR) in the treatment of rectal cancer often are underused out of concern for inadequate distal margins and local failure. The present study addresses whether sphincter-sparing techniques with distal margins < or = 1 cm adversely influence oncological outcome in patients given preoperative chemoradiotherapy. METHODS: Thirty-seven patients with rectal cancer < or = 8 cm from the anal verge were enrolled in the study. Preoperative external beam radiotherapy (5400 Gy) was administered together with continuous infusion of 5-fluorouracil (300 mg/m2/day). Surgical resection was performed in 36 patients with pathological assessment of tumor response and margins. Patients with sphincter-sparing resection and distal margins > 1 cm or < or = 1 cm and those who underwent APR were compared. RESULTS: Thirty-six patients completed preoperative chemoradiotherapy, with successful sphincter-preservation in 28 patients. At a median follow-up of 33 months, there were 12 recurrences overall, which included 11 distant failures and four pelvic failures. Disease-free survival (DFS) was not different between those who had an APR compared with sphincter-sparing resection with distal margins < or = 1 cm. DFS was worse (P < .02) when radial margins were < or = 3 mm compared with > 3 mm. CONCLUSIONS: Sphincter preservation is feasible in more than 75% of patients with tumors < or = 8 cm from the anal verge after preoperative chemoradiotherapy. Sphincter-sparing surgery with distal margins < or = 1 cm can be used without adversely influencing local recurrence or DFS. Limited radial margins (< or = 3 mm), however, are associated with increased disease recurrence.

Activity of Ca2+/calmodulin-dependent protein kinase II following ischemia: a comparison between CA1 and dentate gyrus in a hippocampal slice model.

Both CA1 and dentate gyrus regions of the hippocampal slice exhibit an irreversible loss of synaptic transmission after exposure to in vitro ischemic conditions (buffer without oxygen and glucose). However, after shorter durations of ischemia (8-10 min) the CA1 region shows an irreversible loss of synaptic responses, whereas the dentate gyrus region completely recovers synaptic responses upon reoxygenation. To determine biochemical mechanisms underlying this differential susceptibility, we have examined changes in Ca2+/calmodulin-dependent protein kinase II (CaM-KII) and cyclic AMP-dependent protein kinase activities in homogenates from CA1 and dentate gyrus regions of the hippocampal slice after increasing durations of in vitro ischemia. Time-dependent changes in CaM-KII activities were correlated with changes in electrophysiological responses. CA1 homogenates from slices exposed to 1 min of ischemia showed significant increases in CaM-KII activity, whereas there was no significant change in kinase activity in dentate homogenates after 1 min of ischemia. However, after longer durations of ischemia (5, 10, and 20 min) we found a time-dependent reduction in CaM-KII activity in both CA1 and dentate gyrus regions, whereas no change was detected in cyclic AMP-dependent protein kinase activity. Irreversible depression of CaM-KII activity was seen at shorter durations of ischemia (10 min) in the CA1 region than in dentate region (20 min), which correlated with irreversible effects on synaptic responses. Immunoblot analysis showed that the decrease in CaM-KII activity was not due to degradation of CaM-KII protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiation therapy for skin tumors.

Radiation therapy is an acceptable alternative for the treatment of many malignancies of the skin. Results are gratifying, and long-term sequelae are few. In basal cell and squamous cell carcinomas, radiation is often the best treatment when surgery is expected to cause excessive morbidity or mortality or require extensive reconstruction. Radiation should be considered as first-line treatment for tumors in the midfacial triangle. It is also very acceptable treatment for Kaposi's sarcoma and mycosis fungoides. Radiation can be used for melanoma, although it is not standard first-line therapy. Radiation is probably the treatment of choice for Merkel cell carcinoma.

In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain.

Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.

Failure of 1H nuclear magnetic resonance spectroscopy of blood plasma to detect malignancy.

Water-suppressed proton nuclear magnetic resonance (NMR) of plasma was proposed as a technique for detecting malignant tumors. In that analysis, bloods drawn from cancer patients at the Beth Israel Hospital (BIH; Boston, MA), were easily distinguished from normal subjects by measuring and averaging the proton NMR methyl and methylene line widths of plasma lipoproteins. We collected blood at the Massachusetts General Hospital (MGH), including from normal controls, patients with untreated and treated malignant tumors, and patients with nontumor diseases. The plasma NMR analyses were carried out blind. The code was not broken until all patient charts and pathology records were reviewed, plasma analyses were completed, and patients had been divided into appropriate clinical groups. Analysis of these data showed no differences between the means of the study groups (false-positive and false-negative frequencies 46% and 57%, respectively). An inverse correlation of methyl/methylene line widths with age (P less than .01), and a correlation with nitrate-requiring cardiovascular disease (P less than .05) was, however, evident. This test cannot be validly used to detect malignancy.

Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity.

We have examined the role of Thr-286 autophosphorylation in the autoregulation of Ca2+/calmodulin-dependent protein kinase II. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-286 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.

The influence of estrous cycle and intrastriatal estradiol on sensorimotor performance in the female rat.

The influence of estrous cycle and intrastriatal implants of 17 beta-estradiol (17 beta-estradiol). 17 alpha-estradiol (17 alpha-estradiol) or cholesterol on the number of footfaults made by female rats traversing a narrow suspended beam was investigated. Female rats made fewer footfaults on estrus than on other days of the cycle. This was true when testing occurred during either the light or dark phase of the light:dark cycle. Intrastriatal implants of 30% 17 beta-estradiol for 6 hours resulted in a significant improvement in sensorimotor performance as soon as 4 hours after hormone implant and persisting for days. In contrast, intrastriatal implants of either 30% 17 alpha-estradiol or cholesterol had no influence on performance. The extent of hormone diffusion away from the implant cannula was minimal, and the resulting concentration of 17 beta-estradiol in the striatum was less than 10 pg/mg. It is concluded that estradiol has a direct, stereospecific effect in the striatum that influences performance of a skilled motor act in the female rat.

Impact of improved local control on survival.

Estimates of the gain in survival, if all local failures were eliminated, indicate that many more patients could be cured provided the efficacy of treatment of the primary and regional disease were substantially improved. The expected gain in survival is assumed to be the gain in local control, less the loss due to distant metastases and intercurrent disease among the new local control subjects. The observed incidence of DM among local failure patients may be higher than among local control patients; this excess in incidence of DM is assumed to result from metastases established secondary to the persistent or recurring tumor. A powerful argument that higher local control rates would result in more cured patients is the high incidence of long-term survivors after salvage surgery for local failures. Examples of higher survival associated with more effective local therapy are presented from the literature for medulloblastoma, ependymoma, carcinoma of the oral cavity-oropharynx, carcinoma of the urinary bladder, carcinoma of the prostate and carcinoma of the rectum. For Stage I-II cancer of the breast, the reduction of an already low local failure rate by combining surgery and radiation has a very small impact. For tumors, such as, early stage breast cancer, where the possible decrease in local failure is small and the loss due to DM is high, a demonstrable gain in survival is not likely. The potential increase in number of survivors among the U.S. cancer population, if the primary-regional disease were regularly treated successfully, indicates large gains for patients with cancer of the uterine cervix, oral cavity-oropharynx, ovary, colo-rectum, non-oat cell cancer of lung, prostate cancer, and bladder cancer. These provide powerful bases for aggressive investigation of new approaches to improvement of local-regional therapies.