Mid-term Incidence of Tarsometatarsal Joint Arthrodesis Following Open Reduction With Internal Fixation (ORIF) of Lisfranc Injuries.

Open reduction with internal fixation (ORIF) of Lisfranc injuries are associated with an increased risk for secondary surgery including hardware removal and salvage arthrodesis. In the current literature, rates of salvage arthrodesis vary due to small sample sizes and a low incidence of Lisfranc injuries. There is little evidence to identify specific surgical and patient-related variables that may result in later arthrodesis. The purpose of this study is to determine the rate of tarsometatarsal joint arthrodesis following Lisfranc ORIF in a relatively large sample size. This retrospective review included patients who underwent ORIF for a Lisfranc injury between January 2007 and December 2012. A total of 146 patients met our criteria. Trans-articular fixation was used in 109 (74.6%) patients, 33 (22.6%) received percutaneous fixation and 4 (2.7%) extraarticular fixation. Five out of 120 (4.2%) patients required a salvage arthrodesis for post-traumatic arthritis that had a follow-up greater than 5 y but up to 10 y. The mean age of patients who underwent arthrodesis after ORIF was 24.5 ± 11.95 (16-48) y compared to 40.9 ± 15.8 (16-85) y. Patients who required an arthrodesis also had earlier hardware removal than patients who did not have an arthrodesis, 71.2 ± 28.3 (38-100) days and 131.4 ±101.2 (37-606) days, respectively. Patients who required salvage arthrodesis tended to be younger and hardware was removed earlier compared to those patients who did not require an arthrodesis. Four of the 5 patients who underwent a secondary arthrodesis had a loss of correction after hardware removal.

Patterns of palliative radiotherapy fractionation for brain metastases patients in New South Wales, Australia.

BACKGROUND AND PURPOSE: There is a paucity of studies examining variation in the use of palliative radiation therapy (RT) fractionation for brain metastases. The aim of this study is to assess variation in palliative RT fractionation given for brain metastases in New South Wales (NSW), Australia, and identify factors associated with variation. MATERIALS AND METHODS: This is a population-based cohort of patients who received whole brain RT (WBRT) for brain metastases (2009-2014), as captured in the NSW Central Cancer Registry. A logistic regression model was used to identify factors associated with fractionation type. RESULTS: Of the 2,698 patients that received WBRT, 1,389 courses (51%) were < 6 fractions, 1,050 courses (39%) were 6-10 fractions, and 259 courses (10%) were > 10 fractions. Older patients were more likely to be treated with shorter courses (P < 0.0001). Patients with primary lung cancers were more likely to receive shorter courses compared with other primary cancers (P < 0.0001). Patients without surgical excision were more likely to receive < 6 fractions compared to those who underwent surgical excision. Shorter courses were more likely to be delivered to patients with the most disadvantaged socioeconomic status (SES) compared with patients with the least disadvantaged SES (P < 0.0001). There were significant fluctuations in the proportion of courses using lower number of fractions over time from 2009 to 2014, but no apparent trend (P = 0.02). There was wide variation in the proportion of shorter courses across residence local health districts, ranging from 24% to 69% for < 6 fractions, 21% to 72% for 6-10 fractions, and 4% to 20% for > 10 fractions (P < 0.0001). CONCLUSION: This study has identified significant unwarranted variations in fractionation for WBRT in NSW. Accelerating the uptake of shorter fractionation regimens, if warranted through evidence, should be prioritised to enhance evidence-based care.

Patterns of use of palliative radiotherapy fractionation for bone metastases and 30-day mortality.

BACKGROUND AND PURPOSE: Adoption of single-fraction radiation therapy (SFRT) has not been universal in the palliative treatment of bone metastases, despite evidence supporting its safety and efficacy. The aim of this study was to assess SFRT use for bone metastases in New South Wales (NSW), Australia, and the rate of 30-day mortality (30DM). MATERIALS AND METHODS: This is a population-based cohort of patients who received palliative radiation therapy (RT) for bone metastases (2009-2014), as captured in the NSW Central Cancer Registry. A logistic regression model was used to identify factors associated with fractionation type. The proportion of patients dying within 30-days from treatment start date was calculated. RESULTS: Of the 14,602 courses of palliative RT delivered for bone metastases, 30% were SFRT. SFRT was more likely to be delivered to older patients: ≥80 years (34%) versus < 60 years (28%). Patients with lower socioeconomic status (SES) (35%) were more likely to receive SFRT compared with higher SES (25%). SFRT delivered to patients from outer regional area of residence (34%) were higher compared to those from the major city (29%). The proportion of SFRT delivered to patients with comorbidities ≥2 (34%) was higher than patients with no comorbidity (29%). SFRT was associated with higher 30DM of 21% compared with 11% for multi-fraction RT (MFRT). CONCLUSION: SFRT is underused for the treatment of bone metastases in NSW. This is an impetus to develop tools making SFRT obligatory in this setting unless there is good justification not to.

The population benefit of evidence-based radiotherapy: 5-Year local control and overall survival benefits.

BACKGROUND: To describe the population benefit of radiotherapy in a high-income setting if evidence-based guidelines were routinely followed. METHODS: Australian decision tree models were utilized. Radiotherapy alone (RT) benefit was defined as the absolute proportional benefit of radiotherapy compared with no treatment for radical indications, and of radiotherapy over surgery alone for adjuvant indications. Chemoradiotherapy (CRT) benefit was the absolute incremental benefit of concurrent chemoradiotherapy over RT. Five-year local control (LC) and overall survival (OS) benefits were measured. Citation databases were systematically queried for benefit data. Meta-analysis and sensitivity analysis were performed. FINDINGS: 48% of all cancer patients have indications for radiotherapy, 34% curative and 14% palliative. RT provides 5-year LC benefit in 10.4% of all cancer patients (95% Confidence Interval 9.3, 11.8) and 5-year OS benefit in 2.4% (2.1, 2.7). CRT provides 5-year LC benefit in an additional 0.6% of all cancer patients (0.5, 0.6), and 5-year OS benefit for an additional 0.3% (0.2, 0.4). RT benefit was greatest for head and neck (LC 32%, OS 16%), and cervix (LC 33%, OS 18%). CRT LC benefit was greatest for rectum (6%) and OS for cervix (3%) and brain (3%). Sensitivity analysis confirmed a robust model. INTERPRETATION: Radiotherapy provides significant 5-year LC and OS benefits as part of evidence-based cancer care. CRT provides modest additional benefits.

The Benefits of Providing External Beam Radiotherapy in Low- and Middle-income Countries.

More than half of all cancer diagnoses worldwide occur in low- and middle-income countries (LMICs) and the incidence is projected to rise substantially within the next 20 years. Radiotherapy is a vital, cost-effective treatment for cancer; yet there is currently a huge deficit in radiotherapy services within these countries. The aim of this study was to estimate the potential outcome benefits if external beam radiotherapy was provided to all patients requiring such treatment in LMICs, according to the current evidence-based guidelines. Projected estimates of these benefits were calculated to 2035, obtained by applying the previously published Collaboration for Cancer Outcomes, Research and Evaluation (CCORE) demand and outcome benefit estimates to cancer incidence and projection data from the GLOBOCAN 2012 data. The estimated optimal radiotherapy utilisation rate for all LMICs was 50%. There were about 4.0 million cancer patients in LMICs who required radiotherapy in 2012. This number is projected to increase by 78% by 2035, a far steeper increase than the 38% increase expected in high-income countries. National radiotherapy benefits varied widely, and were influenced by case mix. The 5 year population local control and survival benefits for all LMICs, if radiotherapy was delivered according to guidelines, were estimated to be 9.6% and 4.4%, respectively, compared with no radiotherapy use. This equates to about 1.3 million patients who would derive a local control benefit in 2035, whereas over 615 000 patients would derive a survival benefit if the demand for radiotherapy in LMICs was met. The potential outcome benefits were found to be higher in LMICs. These results further highlight the urgent need to reduce the gap between the supply of, and demand for, radiotherapy in LMICs. We must attempt to address this 'silent crisis' as a matter of priority and the approach must consider the complex societal challenges unique to LMICs.

A Population-based Model of Local Control and Survival Benefit of Radiotherapy for Lung Cancer.

AIMS: To estimate the population-based locoregional control and overall survival benefits of radiotherapy for lung cancer if the whole population were treated according to evidence-based guidelines. These estimates were based on a published radiotherapy utilisation (RTU) model that has been used to estimate the demand and planning of radiotherapy services nationally and internationally. MATERIALS AND METHODS: The lung cancer RTU model was extended to incorporate an estimate of benefits of radiotherapy alone, and of radiotherapy in conjunction with concurrent chemotherapy (CRT). Benefits were defined as the proportional gains in locoregional control and overall survival from radiotherapy over no radiotherapy for radical indications, and from postoperative radiotherapy over surgery alone for adjuvant indications. A literature review (1990-2015) was conducted to identify benefit estimates of individual radiotherapy indications and summed to estimate the population-based gains for these outcomes. Model robustness was tested through univariate and multivariate sensitivity analyses. RESULTS: If evidence-based radiotherapy recommendations are followed for the whole lung cancer population, the model estimated that radiotherapy alone would result in a gain of 8.3% (95% confidence interval 7.4-9.2%) in 5 year locoregional control, 11.4% (10.8-12.0%) in 2 year overall survival and 4.0% (3.6-4.4%) in 5 year overall survival. For the use of CRT over radiotherapy alone, estimated benefits would be: locoregional control 1.7% (0.8-2.4%), 2 year overall survival 1.7% (0.5-2.8%) and 5 year overall survival 1.2% (0.7-1.9%). CONCLUSIONS: The model provided estimates of radiotherapy benefit that could be achieved if treatment guidelines are followed for all cancer patients. These can be used as a benchmark so that the effects of a shortfall in the utilisation of radiotherapy can be better understood and addressed. The model can be adapted to other populations with known epidemiological parameters to ensure the planning of equitable radiotherapy services.

The population benefit of radiotherapy for cervical cancer: local control and survival estimates for optimally utilized radiotherapy and chemoradiation.

PURPOSE: Population benefits of radiotherapy if evidence-based guidelines were routinely followed across the entire population are largely unknown. The aim of this study was to investigate population-based benefits for cervical cancer. METHODS: Overall survival (OS) and local control (LC) benefits were investigated. XRT benefit was defined as the absolute benefit of radiotherapy, over no treatment, for radical indications and defined as the benefit of adjuvant radiation over surgery alone for adjuvant indications. The concurrent chemoradiation (CRT) benefit was the incremental benefit of CRT over XRT. Australian population benefits were modeled using decision trees. Citation databases were systematically queried. Meta-analysis was performed if multiple sources of the same evidence level existed. Robustness of the model assumptions was tested through sensitivity analysis. RESULTS: 53% of all cervix patients had adjuvant or curative radiotherapy indications. 96% were for CRT. The estimated 5-year absolute benefits of optimally utilized radiotherapy alone were: LC: 31% (95% Confidence Interval 29%, 34%), OS: 17% (15%, 18%). These were over and above the contribution of other modalities to outcomes. The incremental 5-year absolute benefits of CRT were: LC 4% (2%, 5%), OS 3% (1%, 5%). In sensitivity analysis, the model was robust. CONCLUSIONS: Optimally utilized radiotherapy provides substantial population OS and LC benefits for cervical cancer. Chemoradiation provides a modest population benefit over XRT. The population-based model was robust.

Estimating the population benefit of radiotherapy: using demand models to estimate achievable cancer outcomes.

The measurement of population benefits is important for priority setting, economic evaluation and quality improvement. It also informs advocacy. In this article, the use of demand models to estimate the achievable benefit of cancer therapy is reviewed. Achievable benefit refers to the treatment benefit achievable under optimal conditions. The population benefit of radiotherapy has been used as an example. Demand models provide a means of estimating the optimal proportion of patients with treatment indications when guidelines are followed. They may be used to estimate achievable benefit. The choice of end point should reflect the range of benefits associated with the treatment of interest. In some cases, further model development is needed if a pre-existing demand model is used. The benefit of treatment for each indication is estimated using a systematic review process. The highest level of evidence is used to define the benefit for each indication. In cases where multiple sources of the same level and quality of evidence exist, a meta-analysis is carried out. Population-based effectiveness data sources are considered, but three major challenges to their use are: (i) generalisability of the observed outcomes, (ii) data resolution and (iii) confounding and bias. The population benefit determined from this process describes the population proportion achieving a benefit due to the use of guideline-based treatment, compared with no use of that treatment. Sensitivity analysis provides a means for modelling the effect of model uncertainties. The predominant uncertainty is most often due to uncertainty in indication proportion. Preference-sensitive treatment decisions are a common example. The described approach to estimating the achievable benefit of cancer therapy is robust to model uncertainties, rapidly adaptable and is transparent. However, estimates rely on the quality of model data sources and may be affected by model assumptions. Models should be developed for a broader range of modalities of cancer therapy and relevant end points.

A comparison of surgical and radiotherapy breast cancer therapy utilization in Canada (British Columbia), Scotland (Dundee), and Australia (Western Australia) with models of "optimal" therapy.

BACKGROUND: Different jurisdictions report different breast cancer treatment rates. Evidence-based utilization models may be specific to derived populations. We compared predicted optimal with actual radiotherapy utilization in British Columbia, Canada; Dundee, Scotland; and Perth, Western Australia. DESIGN: Data were analyzed for differences in demography, tumor, and treatment. Epidemiological data were fitted to published Australian optimal radiotherapy utilization trees and region-specific optimal treatment rates were calculated. Optimal and actual surgery/radiotherapy rates from 2 population-based and 1 institution-based registries were compared for patients diagnosed with breast cancer between 2000 and 2004, and 2002 for British Columbia. RESULTS: Mastectomy rates differed between British Columbia (40%), Western Australia (44%), and Dundee (47%, p<0.01). Radiotherapy rates differed between British Columbia (60%), Western Australia (52%), and Dundee (49%, p<0.01). Actual radiotherapy utilization rates were lower than optimal estimates. Region-specific optimal utilization rates at diagnosis varied from 57% to 71% for radiotherapy and 62% to 64% when taking into account patient preference. Variation was attributed to local differences in demography and tumor stage. CONCLUSIONS: Actual treatment rates varied, and were associated with patterns of care and guideline differences. Actual radiotherapy rates were lower than optimal rates. Differences between optimal and actual utilization may be due to access shortfalls, and patient preference.

Management of skin toxicity during radiation therapy: a review of the evidence.

Acute skin toxicity occurs in the majority of the patients undergoing radical radiotherapy. While a variety of topical agents and dressing are used to ameliorate side effects, there is minimal evidence to support their use. The aims of this study were to systematically review evidence on acute skin toxicity management and to assess the current practices in ANZ. A systematic review of the literature was conducted on studies published between 1980 and 2008. A meta-analysis was performed on articles on clinical trials reporting grade II or greater toxicity. Analyses were divided into breast (the most common site) and other sites. A survey of Radiation Oncology departments across ANZ was conducted to identify patterns of practices and compare these with the published evidence. Twenty-nine articles were reviewed. Only seven articles demonstrated statistically significant results for management of side-effects. These were for topical corticosteroids, hyaluronic acid, sucralfate, calendula, Cavilon cream (3M, St Paul, Minnesota, USA) and silver leaf dressing. Meta-analysis demonstrated statistical significance for the prophylactic use of topical agents in the management acute toxicity. The survey of departments had a low response rate but demonstrated variation in skin care practices across ANZ. A considerable number of these practices were based only on anecdotal evidence. Lack of evidence in the literature for the care of radiation skin reactions was associated with variation in practice. Only a limited number of studies have demonstrated a significant benefit of specific topical agents. There is a need for objective and prospective recording of skin toxicity to collect meaningful comparative data on which to base recommendations for practice.

Dosimetric implications of the addition of 18 fluorodeoxyglucose-positron emission tomography in CT-based radiotherapy planning for non-small-cell lung cancer.

Summary The aim of this study was to assess the impact of F-18 fluorodeoxyglucose-positron emission tomography (FDG-PET) CT on radiotherapy planning parameters for patients treated curatively with radiotherapy for non-small-cell lung cancer (NSCLC). Five patients with stages I-III NSCLC underwent a diagnostic FDG-PET CT (dPET CT), planning FDG-PET CT (pPET CT) and a simulation CT (RTP CT). For each patient, three radiation oncologists delineated a gross tumour volume based on RTP CT alone, and fused with dPET CT and pPET CT. Standard expansions were used to generate PTVs, and a 3D conformal plan was created. Normal tissue doses were compared between plans. Coverage of pPET CT PTV by the plans based on RTP CT and dPET CT was assessed, and tumour control probabilities were calculated. Mean PTV was similar between RTP CT, dPET CT and pPET CT, although there were significant inter-observer differences in four patients. The plans, however, showed no significant differences in doses to lung, oesophagus, heart or spinal cord. The RTP CT plan and dPET CT plan significantly underdosed the pPET PTV in two patients with minimum doses ranging from 12 to 63% of prescribed dose. Coverage by the 95% isodose was suboptimal in these patients, but this did not translate into poorer tumour control probability. The effect of fused FDG-PET varied between observers. The addition of dPET and pPET did not significantly change the radiotherapy planning parameters. Although FDG-PET is of benefit in tumour delineation, its effect on normal tissue complication probability and tumour control probability cannot be predicted.

Use of chemotherapy at end of life in oncology patients.

BACKGROUND: Appropriately timed cessation of chemotherapy is integral to patient's quality of life. We evaluate the use of and associated factors with chemotherapy at the end of life. METHODS: A review of deceased oncology patients treated with palliative intent from April 2005 over 2 years at two cancer centres was carried out. Chi-square tests of patient demographics, cancer and chemotherapy variables were carried out to determine associations with commencing chemotherapy and continuation within 2 and 4 weeks of death. Multivariate analyses were carried out with significant factors to determine their independent effect. RESULTS: Seven hundred and forty-seven patients died during this period; median age 67 years (range 20-96); female 44%. Three hundred and ninety-eight (53%) received chemotherapy: 18% and 8% within 4 and 2 weeks of death, respectively. Younger age (P < 0.01), cancer type (P < 0.01) and chemosensitivity of the tumour (P < 0.01) were predictors for commencing chemotherapy in multivariate analysis. Treating doctor predicted for chemotherapy in the 4 weeks before death (<0.05), but none of the variables predicted for chemotherapy in the last 2 weeks of life. CONCLUSIONS: Younger age, tumour type and chemosensitivity are important predictors of patients receiving palliative chemotherapy. Individual clinician was the only predictor for continuing chemotherapy in the last 4 weeks of life.

Polyclonal expansion of peripheral gamma delta T cells in human Plasmodium falciparum malaria.

Plasmodium falciparum malaria in humans is associated with an increase in the percentage and absolute number of gamma delta T cells in the peripheral blood. This increase begins during the acute infection phase and persists for at least 4 weeks during convalescence. In the present study, 25 to 30% of the gamma delta T cells expressed HLA-DR antigens in vivo and in some patients they proliferated in response to further stimulation by purified human interleukin 2 in vitro. However, there was no in vitro proliferative response to various malarial antigens, including a 75-kDa heat shock protein and a 72-kDa glucose-regulated protein of P. falciparum during the acute infection phase. Cytofluorographic studies showed that although an increase of V delta 1- gamma delta T cells was largely responsible for the expansion of the total number of gamma delta T cells, there was also a proportional increase in V delta 1+ cells. These results were confirmed with anchored PCR and by DNA sequencing to characterize at the molecular level the set of T-cell receptor (TCR) delta mRNAs expressed in the peripheral blood of two patients with high levels of gamma delta T cells. In each case, most of the TCR delta mRNA transcripts corresponded to nonproductively rearranged delta genes (unrearranged J delta or near J delta spliced to C delta). In those sequences which did represent productively rearranged genes, most of the transcripts originated from a V delta 2/J delta 1 joining, as in normal individuals. A minority of transcripts originated from a V delta 1/J delta 1 rearrangement, and one originated from a V alpha 4/J delta 1 rearrangement. Polyclonal activation of gamma delta T cells was inferred from the extensive junctional diversity seen in the delta mRNAs analyzed. Expansion of a heterogeneous set of both V delta 1(-)- and V delta 1(+)-bearing T cells suggests that the elevated levels of gamma delta T cells seen during acute P. falciparum malaria arose from immune responses to multiple distinct parasite antigens or unidentified host factors.

Effects of a newly synthesized K+ channel opener, Y-26763, on noradrenaline-induced Ca2+ mobilization in smooth muscle of the rabbit mesenteric artery.

1. The mechanisms underlying the vasodilatation induced by (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2, 2-dimethyl-2H-1-benzopyran-3-ol (Y-26763) were investigated by measuring membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and isometric force in smooth muscle cells of the rabbit mesenteric artery. 2. Y-26763 (0.03-1 microM) concentration-dependently hyperpolarized the membrane and glibenclamide (1-10 microM) inhibited this hyperpolarization. Noradrenaline (NA, 10 microM) depolarized the membrane and generated spike potentials. Y-26763 (1 microM) inhibited these NA-induced electrical responses. 3. In thin smooth muscle strips in 2.6 mM Ca2+ containing (Krebs) solution, 10 microM NA produced a large phasic, followed by a small tonic increase in [Ca2+]i and force with associated oscillations. In Ca(2+)-free solution (containing 2 mM EGTA), NA produced only phasic increases in [Ca2+]i and force. In ryanodine-treated strips, NA could not produce the phasic increases in [Ca2+]i and force even in the presence of 2.6 mM Ca2+, suggesting that ryanodine functionally removes the NA-sensitive intracellular storage sites. 4. Nicardipine (1 microM) partly inhibited the NA-induced tonic increases in [Ca2+]i and force but had no effect on either the resting [Ca2+]i or the NA-activated phasic increases in [Ca2+]i and force. By contrast, Y-26763 (10 microM) lowered the resting [Ca2+]i and also inhibited both the phasic and the tonic increases in [Ca2+]i and force induced by NA. All these actions of Y-26763 were inhibited by glibenclamide (10 microM). 5. In ryanodine-treated strips, nicardipine partly, but Y-26763 completely inhibited the NA-induced increases in [Ca2+]i, suggesting that Y-26763 inhibits both the nicardipine-sensitive and -insensitive Ca2+ influxes activated by NA. Y-26763 attenuated the phasic increase in [Ca2+]i and force in a Ca(2+)-free solution containing 5.9 mM K+, but not in one containing 50 mM K+, suggesting that Y-26763 inhibits NA-induced Ca2+ release, probably as a result of its membrane hyperpolarizing action. 6. In Beta-escin-skinned strips, Y-26763 (10 MicroM) had no effect on either the NA-induced Ca2+ release or the Ca2+-tension relationship in the presence and absence of NA (10 MicroM) with guanosine 5'-triphosphate(GTP, 10 MicroM), suggesting that Y-26763 has no direct action on either NA-induced Ca2+ release or the contractile proteins.7. It is concluded that Y-26763 inhibits NA-activated Ca2+ release and Ca2+ influx and thus inhibits the NA-contraction. Y-26763 also lowers the resting [Ca2+]i through an inhibition of the nicardipine insensitive Ca2+ influx. These actions of Y-26763 may be linked with the membrane hyperpolarization it produces by activation of the ATP-sensitive K+ channels.

Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery.

To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.

Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.

Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria.