Angiogenic effects despite limited cell survival of bone marrow-derived mesenchymal stem cells under ischemia.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

siRNA silencing of keratinocyte-specific GFP expression in a transgenic mouse skin model.

Small interfering RNAs (siRNAs) can be designed to specifically and potently target and silence a mutant allele, with little or no effect on the corresponding wild-type allele expression, presenting an opportunity for therapeutic intervention. Although several siRNAs have entered clinical trials, the development of siRNA therapeutics as a new drug class will require the development of improved delivery technologies. In this study, a reporter mouse model (transgenic click beetle luciferase/humanized monster green fluorescent protein) was developed to enable the study of siRNA delivery to skin; in this transgenic mouse, green fluorescent protein reporter gene expression is confined to the epidermis. Intradermal injection of siRNAs targeting the reporter gene resulted in marked reduction of green fluorescent protein expression in the localized treatment areas as measured by histology, real-time quantitative polymerase chain reaction and intravital imaging using a dual-axes confocal fluorescence microscope. These results indicate that this transgenic mouse skin model, coupled with in vivo imaging, will be useful for development of efficient and 'patient-friendly' siRNA delivery techniques and should facilitate the translation of siRNA-based therapeutics to the clinic for treatment of skin disorders.

Minimal-preparation abdomino-pelvic CT in frail and elderly patients: prognostic value of colonic and extracolonic findings.

AIM: To examine the overall survival of patients who had had been referred for minimal preparation abdomino-pelvic computed tomography (MPCT), and to assess the prognostic value of the colonic and extracolonic findings detected. METHODS AND MATERIALS: The survival of a cohort of 1029 elderly and frail patients, with clinical symptoms and signs suspicious for colorectal cancer (CRC), who had undergone MPCT between 1995 and 1998 was investigated. Univariate and multivariate survival analyses were undertaken according to the presence of CRC and extracolonic abnormalities (ECA). RESULTS: The median age of the 1029 patients was 79.4 years. The overall median survival following MPCT was 5.4 years; and 6.6 years if no abnormality was detected. On multivariate analysis, age, sex, CRC status, and number of ECAs were significant factors in overall survival. Median survival for those with confirmed CRC [n=91 (prevalence, 8.8%)] was 1.1 years, compared with 5.9 years without CRC (p<0.0001); and 2.4 years for those with one or more ECA [n=245 (prevalence, 23.8%)], compared with 6.1 years without ECA (p<0.0001). Survival was progressively shorter for increasing numbers of ECAs; and shorter for previously unknown non-CRC malignancies (n=24) compared with CRC (p<0.0001). CONCLUSIONS: MPCT appears to have prognostic potential in this patient population, with significant reductions in survival if a CRC or ECA is detected. The detection of ECA would appear to have at least as important an impact on the usefulness of the examination as the detection of CRC.

Clinical characteristics of an anatomical hand index measured in patients with rheumatoid arthritis as a potential outcome measure.

OBJECTIVES: To determine the clinical characteristics of an anatomical hand index previously reported as a potential measure of joint deformity and outcome in patients with rheumatoid arthritis. METHODS: The hand index (open hand span - closed hand span/lateral height of the hand) was measured in a cross-sectional study of 145 out-patients with rheumatoid arthritis with disease durations 0-55 yr. Subsets of patients were restudied at mean follow-ups of approximately 9 months and 4 yr. RESULTS: The hand index fell gradually with disease duration. Correlations were demonstrated with the Sharp index (r = - 0.39, P = 0.000) and to a lesser extent with disease activity score (r = - 0.28, P = 0.001). At 260 +/- 115 days the hand index worsened by 0.09 units (P = 0.09, NS). At 51.6 +/- 5.4 months the index showed a fall from 1.96 +/- 0.73 to 1.61 +/- 0.65 (P = 0.000). During the same interval the Sharp index increased from 60 +/- 68 to 80 +/- 71 (P = 0.000). CONCLUSIONS: Measurement of simple hand dimensions can demonstrate worsening of hand deformity with time in patients with rheumatoid arthritis. We suggest that more sophisticated analysis of digital hand images, as used in our original study, might yield additional information and increase the sensitivity of an anatomical hand index as an outcome measure in rheumatoid arthritis.

Extracolonic findings in patients undergoing abdomino-pelvic CT for suspected colorectal carcinoma in the frail and disabled patient.

AIM: The aims of this study were to evaluate the extracolonic findings identified in patients undergoing minimal preparation abdomino-pelvic CT in place of barium enema or colonoscopy for the detection of possible colorectal carcinoma. MATERIALS AND METHODS: The CT technique involved helical acquisition (10 mm collimation, 1.5 pitch) following 2 days of preparation with oral contrast medium only. Extracolonic findings were evaluated in the light of subsequent follow-up and accuracy. The evaluation included assessment of the potential contribution of the extracolonic finding(s) to staging the cancer in the subset of patients who had colorectal carcinoma, and to account for the patients' presenting symptoms and signs in the remaining patients. RESULTS: A total of 344 extracolonic findings were detected in 261 CT examinations, from amongst a total of 1077 cases (24%). Extracolonic findings were potentially important in staging in 32 of the 98 (33%) cases subsequently found to have colorectal cancer. There were 284 extracolonic findings amongst the 221 cases who proved not have colorectal cancer. One hundred and twenty-four (44%) of these 284 findings were actively followed up by clinicians, and 33 (12%) ultimately had a surgical intervention. Fifty-six percent (160/284) of the findings were determined to be correct (by further investigation, autopsy, and/or clinical follow-up); the remainder were incorrect or indeterminate (n = 56) or had no follow-up (n = 68). The commonest extracolonic findings were focal liver lesions (found in 42/1077, 4%) and abdominal aortic aneurysms (31/1077, 3%). Twenty-four (24/1077, 2%) previously unknown extracolonic malignancies were detected. Ten percent (106/1077) of the patients had extracolonic findings that could potentially have accounted for their presenting symptoms. CONCLUSIONS: CT has the added benefit, compared with colonoscopy and barium enema, of not just evaluating the colon but also of detecting extracolonic abnormalities. Such findings may be useful in staging the cancer, may explain the patient's presenting symptoms, and may detect other potentially serious disorders.

Evaluation of CT in identifying colorectal carcinoma in the frail and disabled patient.

Frail and physically or mentally disabled patients frequently have difficulty in tolerating formal colonic investigations. The aims of this study were to evaluate the accuracy of minimal-preparation CT in identifying colorectal carcinoma in this population and to determine the clinical indications and radiological signs with the highest yield for tumour. The CT technique involved helical acquisition (10-mm collimation, 1.5 pitch) following 2 days of preparation with oral contrast medium only. The outcome of 4 years of experience was retrospectively reviewed. The gold standards were pathological and cancer registration records, together with colonoscopy and barium enema when undertaken, with a minimum of 15 months follow-up. One thousand seventy-seven CT studies in 1031 patients (median age 80 years) were evaluated. CT correctly identified 83 of the 98 colorectal carcinomas in this group but missed 15 cases; sensitivity and specificity (with 95% confidence interval) 85% (78-92%) and 91% (90-93%), respectively. Multivariate analysis identified: (a) a palpable abdominal mass and anaemia to be the strongest clinical indications, particularly in combination (p<0.0025); and (b) lesion width and blurring of the serosal margin of lesions to be associated with tumours (p<0.0001). Computed tomography has a valuable role in the investigation of frail and otherwise disabled patients with symptoms suspicious for a colonic neoplasm. Although interpretation can be difficult, the technique is able to exclude malignancy with good accuracy.

Bilateral coronoid hyperplasia in two brothers.

Coronoid hyperplasia is a rare condition of unknown aetiology that can occur in both unilateral and bilateral forms. Without radiographic investigation the diagnosis is often missed. Researchers have postulated a familial form of inheritance. This study reports the occurrence of coronoid hyperplasia in two brothers. The parents were unaffected and there are no other siblings. The diagnosis was confirmed with the aid of panoramic radiographs and axial computed tomographic scans with para-sagittal reconstructions which demonstrated enlargement of the coronoid processes and in one case impingement against the zygomatic bone. One brother was successfully treated with a unilateral intra-oral coronoidectomy whilst the other was unsuccessfully treated with a bilateral intra-oral coronoidectomy.

Caecal carcinomas in the elderly: useful signs in minimal preparation CT.

OBJECTIVE: Frail, elderly and immobile patients frequently have difficulty in tolerating formal colonic investigations. Caecal tumours may account for up to 35% of colonic tumours. Barium enema and colonoscopy have limitations in assessing this region. The aims of this study were to evaluate the accuracy of a minimal preparation CT technique (merely with prolonged oral contrast medium) in identifying caecal carcinomas and to determine helpful radiological signs. MATERIALS AND METHODS: The CT technique involved helical acquisition following 2 days of preparation with oral contrast medium. The outcome of 4 years' experience (1995-1998) was reviewed. The gold-standards were pathological and cancer registration records, together with colonoscopy and barium enema where available, with a minimum of 15 months' follow-up. RESULTS: CT correctly identified 27 of 30 caecal carcinomas, and missed three, in a total of 1077 CT studies in 1031 patients (median age 80 years). There were also 21 false-positive cases in which CT incorrectly raised the possibility of a caecal tumour. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were 90%, 98%, 99% and 56%, respectively. Serosal margin blurring, tumour length, presence of abnormal peri-colic fat and terminal ileal wall thickening were identified as useful radiological signs. CONCLUSIONS: Minimal preparation CT is able to identify caecal carcinomas with fair accuracy. Such evaluation may become important given the increasing population age and evidence of a 'proximal shift' in the site of colonic tumours in the elderly.

Histopathological correlates of abnormal pericolic fat on CT in the assessment of colorectal carcinoma.

The aim of this study was to assess the pathological significance of abnormal pericolic fat shown by CT in the context of colorectal carcinoma. CT and histopathological findings of 63 resected colorectal carcinomas were retrospectively reviewed. CT examinations were assessed by two observers for the presence or absence of abnormal pericolic fat (typically linear or nodular opacities) at tumour sites. Specimens were reviewed histopathologically for depth of tumour invasion, extramuscular tissue reaction, and number and largest size of tumour-involved and tumour-free lymph nodes. The sensitivity, specificity, positive predictive value and negative predictive value of pericolic fat in identifying extension of tumour infiltration beyond the muscle coat were 79% (42/53), 33% (2/6), 91% (42/46) and 15% (2/13), respectively. Despite these indicators of efficacy, the association between the presence of pericolic fat abnormality on CT and extramuscular extension of tumour (infiltration and/or nodal disease) or tissue reaction alone or in combination did not reach statistical significance (p>0.3 in all cases). Abnormal ("misty" or "mucky") pericolic fat in the assessment of colorectal cancer on CT is not a precise indicator of extramuscular extension of tumour, as it cannot clearly distinguish between tumour infiltration and tissue reaction beyond the muscle coat, or pericolic nodal involvement. However, it is a very helpful CT sign that may draw attention to the presence and site of a potential colonic abnormality.

Urinary iodine and thyroid status of New Zealand residents.

OBJECTIVES: The aim of this project was to assess the clinical significance of our low iodine excretions in terms of thyroid hormone status and thyroid volume in an adult population in a low soil iodine area of the South Island of New Zealand. DESIGN AND SETTING: Two-hundred and thirty-three residents of Otago, New Zealand collected two 24 h urine samples for assessment of iodine status. Thyroid status was determined from serum total T(4), TSH and thyroglobulin, and thyroid volumes. Relationships between urinary iodide excretion and measures of thyroid status were determined and subjects were allocated to one of three groups according to low, medium and high iodide excretion, for comparison of thyroid hormones and thyroid volumes. RESULTS: Significant correlations were found for relationships between measures of urinary iodide excretion and thyroid volume and thyroglobulin. Multiple regression analysis of data for subjects divided into three groups according to 24 h urinary iodide excretion (<60, 60-90; >90 microg iodide/day) or iodide/creatinine ratio (<40; 40-60; >60 microg/g Cr) showed significant differences in thyroid volume (P=0.029; P=0.035, respectively) and thyroglobulin (P=0.019; P=0.005, respectively) among the groups. CONCLUSIONS: The results of this study confirm the low iodide excretions of Otago residents, and indicate that the fall in iodine status is being reflected in clinical measures of thyroid status, including enlarged thyroid glands and elevated thyroglobulin. Our observations suggest the possible re-emergence of mild iodine deficiency and goitres in New Zealand. This situation is likely to worsen should iodine intakes continue to fall and continued monitoring of the situation is imperative.

Tryptophan fluorescence of yeast actin resolved via conserved mutations.

Actin contains four tryptophan residues, W79, W86, W340, and W356, all located in subdomain 1 of the protein. Replacement of each of these residues with either tyrosine (W79Y and W356Y) or phenylalanine (W86F and W340F) generated viable proteins in the yeast Saccharomyces cerevisiae, which, when purified, allowed the analysis of the contribution of these residues to the overall tryptophan fluorescence of actin. The sum of the relative contributions of these tryptophans was found to account for the intrinsic fluorescence of wild-type actin, indicating that energy transfer between the tryptophans is not the main determinant of their quantum yield, and that these mutations induce little conformational change to the protein. This was borne out by virtually identical polymerization rates and similar myosin interactions of each of the mutants and the wild-type actin. In addition, these mutants allowed the dissection of the microenvironment of each tryptophan as actin undergoes conformational changes upon metal cation exchange and polymerization. Based on the relative tryptophan contributions determined from single mutants, a triple mutant of yeast actin (W79) was generated that showed small intrinsic fluorescence and should be useful for studies of actin interactions with actin-binding proteins.

Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations.

The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to alpha-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10 degrees C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin-actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an alpha-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in alpha-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype.

Hemifacial atrophy with bilateral short roots.

We present a case in which the patient had both bilateral shortening of the roots, and hemifacial atrophy. As far as we know, this combination has not been described before.

Nonspecific weak actomyosin interactions: relocation of charged residues in subdomain 1 of actin does not alter actomyosin function.

Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individual clusters of acidic residues in mediating actomyosin weak-binding states in the cross-bridge cycle. Past studies have established a functional role for three distinct pairs of charged residues within this region of yeast actin (D2/E4, D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same impairment in weak actomyosin interaction and in its function. However, the specificity of myosin interaction with these sites has not yet been addressed. To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged residues (alanines) and a pair of acidic residues was inserted at the N-terminus, maintaining the overall charge density of subdomain 1. Using the in vitro motility assays, we found that the sliding and force generation properties of these mutant actins were identical to those of wild-type actin. Similarly, actin-activated ATPase activities of the mutant and wild-type actins were also indistinguishable. Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin. These results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function.

Clinical outcomes of the Otago-Southland Breast Cancer Screening Programme 1991-1996.

AIM: To document the clinical outcome of the Otago-Southland Breast Cancer Screening Programme through its first two rounds of screening, from 1991-1996. METHODS: Review and analysis of clinical and pathological records. RESULTS: In the first round of screening, 13,876 women were screened, giving 75% uptake; 12.2% were referred for assessment and 126 cancers detected, 9.1 per thousand women screened. For the 9946 incidence screens in the second round, 3.9% of women screened were referred to assessment and 50 cancers detected, 5.0 per thousand women screened. The uptake and cancer detection rates exceed the targets and exceed other published results; the size distribution of the cancers detected was comparable to the Swedish two-counties study, showing that the results should produce an ultimate mortality reduction. The referral rate to assessment was higher than expected in the first round of screening, but within the targeted range in the second round. The benign to malignant ratio for all biopsies was 1.4:1 for the prevalence screen of the first round and 1.2:1 for the incidence screens in the second round, both exceeding the targets set. CONCLUSIONS: The results show that the uptake and clinical results of the programme exceed expectations and that a large number of small invasive tumours have been successfully detected. These results are comparable to the best of overseas studies, and give confidence that mortality reductions will ultimately occur.

Multiple functions for actin during filamentous growth of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is dimorphic and switches from a yeast form to a pseudohyphal (PH) form when starved for nitrogen. PH cells are elongated, bud in a unipolar manner, and invade the agar substrate. We assessed the requirements for actin in mediating the dramatic morphogenetic events that accompany the transition to PH growth. Twelve "alanine scan" alleles of the single yeast actin gene (ACT1) were tested for effects on filamentation, unipolar budding, agar invasion, and cell elongation. Some act1 mutations affect all phenotypes, whereas others affect only one or two aspects of PH growth. Tests of intragenic complementation among specific act1 mutations support the phenotypic evidence for multiple actin functions in filamentous growth. We present evidence that interaction between actin and the actin-binding protein fimbrin is important for PH growth and suggest that association of different actin-binding proteins with actin mediates the multiple functions of actin in filamentous growth. Furthermore, characterization of cytoskeletal structure in wild type and act1/act1 mutants indicates that PH cell morphogenesis requires the maintenance of a highly polarized actin cytoskeleton. Collectively, this work demonstrates that actin plays a central role in fungal dimorphism.

Once is enough--why some women do not continue to participate in a breast cancer screening programme.

AIM: To assess the reasons why many women who have been screened once in a breast screening programme decline an invitation for further screening. METHODS: Telephone interview survey of a sample of such women; for questions relating to their experience of previous mammography, comparison to data on a representative sample of first screen attendees. The subjects were women who had attended the first round of the Otago-Southland breast cancer screening programme in 1991-1994, who were eligible for re-screening but had been rescreened; age range 50-62. RESULTS: From programme records, 86% of women who were eligible for a second screen accepted it. Of the women not recorded as having had a second screen, some had attended for a second screen; some had not been invited until they had become age ineligible and some had received no invitation for re-screening. Of women who had received and declined an invitation for re-screening (n = 81), the major reason (46%) was their previous mammogram being painful. Other factors contributing were illness in themselves or their spouse, practical difficulties arranging time and negative experiences with staff in the previous mammography, although these related to relatively few women. A few women thought mammography would be of no benefit, and a few thought re-screening was unnecessary because their first mammography had been normal, or because they practise self-examination. CONCLUSIONS: Ensuring that all women eligible for further screening do get invited could substantially increase the re-screening rate. Even women who have declined previous invitations should be offered further invitations, as a substantial proportion with to be screened. Flexible and convenient appointment times are the main modifiable logistic issue. The major factor influencing non-participation with further screening is a painful experience of mammography. Innovative approaches, either to reduce the pain or to reduce the impact of the pain on the woman's attitude to re-screening, should be trialed.

The general practitioner's role in breast cancer screening: a survey in Otago-Southland.

AIMS: To study the experience of general practitioners in Otago and Southland with the existing breast cancer screening programme and the reviews on future programmes. METHODS: A questionnaire was sent to all 210 general practitioners in Otago and Southland in June 1996. RESULTS: The response rate was 71%. All the 141 respondents except one encouraged eligible women to take part in the programme; this was done mainly during individual doctor-patient consultations, by pamphlets and posters, and in the work of the practice nurse. Ten percent of practitioners had a practice-based recall system for breast cancer screening. Seventy-five percent of general practitioners currently provide a list of eligible women to the programme, and of these, 52% check the list to exclude ineligible women. Only 24% of practitioners supplying a patient list to the programme reported that a patient had ever requested that their name be excluded from the list. Twenty-five percent of general practitioners providing lists had a notice in the waiting room stating that. Of those who did not provide lists, concerns about logistics, ethical issues and cost were raised, although 40% of these general practitioners intended to provide lists in the future. In a future programme, 57% of general practitioners felt they should be paid for supplying lists defined by age only and 82% felt they should be paid for supplying a list of women eligible by both age and medical history. Most general practitioners felt that general practitioner lists were the preferred source for invitations to the breast screening programme and that general practitioners had an important part in any future programme. Screening at the ages 50-64 (as currently proposed) is supported by 95% of general practitioners; in addition, 64% supported screening at ages 65-69. Only a minority of general practitioners supported screening at ages 40-49 or ages 70-74. Most general practitioners would offer screening to women under age 50 with either a strong or a weak family history, or even with a past history of a fibroadenoma. CONCLUSIONS: These results show that almost all general practitioners support breast cancer screening programmes and feel that they have an important role in future programmes. The majority support extension of the programme to ages 65-69, but not to ages 40-49. The majority support screening women with individual risk factors at ages under 50, although their responses show that better information on the importance of different risk factors is required.