Cash outs during in-play sports betting: Who, why, and what it reveals.

Cashing out is a popular feature of modern 'in-play' sports betting that allows sports bettors to withdraw a bet before the sporting event on which the bet was placed is finalized. Previous studies have shown that use of the cash out feature is positively related to problem gambling symptomatology. However, little is known about demographic and psychological characteristics of in-play sports bettors who use the cash out feature, or their motivations for use. To fill this knowledge gap, we recruited 224 adults (18 + years) from Ontario who engaged in in-play sports betting in the past three months. Participants completed self-report measures of psychological and gambling-related variables. Participants also provided qualitative responses for their motivations for using the cash out feature. Approximately half (51.8 %) of the participants reported using the cash out feature. No statistically significant demographic differences were found between participants who used and did not use the cash out feature. Participants who used the feature (compared to those who did not) reported higher problematic alcohol and cannabis use, feelings of depression, anxiety, and stress, and were motivated to gamble to make money. The primary reasons for cashing out were to access money immediately, to cut losses, and because cashing out felt like a less risky option. The current findings shed light on underlying psychological vulnerabilities associated with individuals who use the cash out feature, which can inform initiatives to reduce the harms associated with this popular feature of sports betting.

Oral Health Advice for Looked-After-Children: A pilot care pathway in Buckinghamshire, UK.

INTRODUCTION: Looked-After-Children (LAC) frequently are more likely to have untreated dental caries, periodontal diseases or dental trauma (McMahon et al., 2018). The COVID-19 pandemic reduced the availability of dental appointments, including for LAC. This initiative piloted the inclusion of a dental pathway into the existing LAC care pathway in Buckinghamshire. The key principle was providing oral health messaging around maintaining good oral health and preventing disease in these children by training those involved with their care. METHODS: A working group was convened, which included dental public health, clinical and training expertise. A care pathway was developed with resources drawn from existing programmes including mini Mouth Care Matters (mMCM). The care pathways were designed to identify children who needed care urgently due to pain or discomfort, signpost them to the relevant/most appropriate providers and provide oral health improvement advice for everyone. Local partners were engaged to ensure that the processes and training were appropriate. A pilot training session was then carried out for a range of staff engaged in the care of LAC within Buckinghamshire. RESULTS: The pilot dental pathway, launched in March 2022, initiative was welcomed by all stakeholders, including dental commissioners. The pilot training session received positive feedback from participants, with requests for more sessions. Training sessions were subsequently translated into video sessions, accessible when needed, for new staff or as refresher sessions. CONCLUSION: There are opportunities to reduce future inequalities for these children by inculcating positive behaviours early in their care journey. This will reduce their need for care. Identifying and implementing the most appropriate initiatives requires collaboration and commitment from all stakeholders.

[The in vitro effect of antiseptics on epithelial cells of human cornea and conjunctiva]. / Vozdeistvie antiseptikov na epitelial'nye kletki rogovitsy i kon"yunktivy cheloveka in vitro.

Effective and safe antiseptic eye preparations are necessary for prevention and treatment of infectious and inflammatory eye diseases. PURPOSE: in vitro evaluation of the effect of antiseptic eye drops on corneal and conjunctival epithelial cells. MATERIAL AND METHODS: Antiseptic eye drops «Bactavit¼, «Vitabact¼ and «Ocomistin¼ were the object of the study. Immortalized human corneal epithelial cell lines (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) were used as the test systems. The viability of the cells was assessed by their metabolic activity and morphology using the MTT test and phase-contrast microscopy. RESULTS: Antiseptic eye drops belonging to different groups of chemical compounds induced cytotoxic effects on the cells of corneal epithelium (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) of varying degrees, leading to morphological and functional changes in those cells. CONCLUSION: The study confirms the possibility of using cultured cells for the in vitro comparative assessment of the cytotoxic effect of antiseptic ophthalmic agents.

Reactivation of mutant p53 by a dietary-related compound phenethyl isothiocyanate inhibits tumor growth.

Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53(R175), one of the most frequent 'hotspot' mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53(R175) mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53(R175) mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53(R175) mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53(R175) and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

Brexit and dentistry.

On 23 June 2016, eligible UK voters will be asked to decide whether to vote in the EU referendum. The EU impacts on our daily lives in more ways than many people realise. Dentistry is affected by EU legislation. Examples include the movement of dental professionals, the import of dental equipment and materials, as well as health and safety legislation. Many more EU dentists and DCPs come to the UK to work than vice versa. These numbers have increased markedly since 2004. The result of the vote may affect how dentistry operates in the UK in future years. In addition, a vote to stay would not necessarily prevent change. There are attempts underway to increase the ease by which professionals can work in other member states, especially on a temporary basis. This too is likely affect dentistry at some point. Workforce planners and policy makers should factor in the impact of the EU in future dental policy.

Radiotherapy for oncologic emergencies on weekends: examining reasons for treatment and patterns of practice at a Canadian cancer centre.

PURPOSE: Radiotherapy for oncologic emergencies is an important aspect of the management of cancer patients. These emergencies-which include malignant spinal cord compression, brain metastases, superior vena cava obstruction, and uncontrolled tumour hemorrhage -may require treatment outside of hospital hours, particularly on weekends and hospital holidays. To date, there remains no consensus among radiation oncologists regarding the indications and appropriateness of radiotherapy treatment on weekends, and treatment decisions remain largely subjective. The main aim of the present study was to document the incidence and indications for patients receiving emergency treatment on weekends or scheduled hospital holidays at a single institution. The secondary aim was to investigate the compliance of such treatment with the institution's quality assurance policies, both local and provincial. METHODS: From September 1, 2002, to September 30, 2004, patients being treated over weekends (defined as commencing at 6 pm on a Friday and concluding at 8 am of the next scheduled workday) and hospital holidays were retrospectively identified using the Oncology Patient Information System scheduling module. Relevant patient data-including patient age, sex, primary cancer site, specific radiation field, rationale for treatment, referring hospital, total treatment dose, radiation dose fractionation, inpatient or outpatient status, and duration of treatment-were collected and subsequently analyzed. Comparison to local policy was performed subjectively. RESULTS: Over the 2-year period, 161 patients were prescribed urgent radiotherapy over a weekend or on a hospital holiday. Of this cohort, 68% were treated on both Saturday and Sunday, 22% on Saturday alone, and 10% on Sunday alone. Most patients presented with lung (31%), prostate (18%), and breast cancer (17%). The top reasons for referral for emergency weekend treatment included spinal cord compression (56%), brain metastases (15%), and superior vena cava obstruction (6%). Most of the indications for treatment generally followed the quality assurance policies implemented both locally and provincially. CONCLUSIONS: Patients treated over a weekend or on a hospital holiday were generally found to be treated with appropriate intent. Most treatment indications within this study both complied with provincial policy and showed a pattern of care similar to that seen in other studies in the literature. Local policy appears to be robust; however, policy improvements may allow for more cohesiveness across radiation oncologists in patterns of care in this important group of patients. Comparisons with practice at other institutions would be valuable and also a key step in developing sound guidelines for all members of the radiotherapy community to follow.

Quality of life measurement in cancer patients receiving palliative radiotherapy for symptomatic lung cancer: a literature review.

Approximately 27% of North American cancer deaths are attributable to cancer of the lung. Many lung cancers are found at an advanced stage, rendering the tumours inoperable and the patients palliative. Common symptoms associated with palliative lung cancer include cough, hemoptysis, and dyspnea, all of which can significantly debilitate and diminish quality of life (QOL). In studies of the effects of cancer therapies, the frequent evaluative endpoints are survival and local control; however, it is imperative that clinical trials with palliative patients also have a QOL focus when a cure is unattainable. We conducted a literature review to investigate the use of QOL instrument tools in trials studying QOL or symptom palliation of primary lung cancer or lung metastases through the use of radiotherapy. We identified forty-three studies: nineteen used a QOL tool, and twenty-four examined symptom palliation without the use of a QOL instrument. The European Organization for Research and Treatment of Cancer (eortc) QLQ-C30 survey was the most commonly used QOL questionnaire (in thirteen of twenty trials). Of those thirteen studies, eight also incorporated the lung-specific QOL survey eortc QLQ-LC13 (or the eortc QLQ-LC17). A second lung-specific survey, the Functional Assessment of Cancer Therapy-Lung (fact-L) was used in only two of the twenty trials. In total, only ten of forty-three trials (23%) used a lung-specific QOL tool, suggesting that QOL was of low priority as an endpoint and that measures created for lung cancer patients are underused. We encourage investigators in future trials to include specific QOL instruments such as the eortc QLQ-LC13 or the fact-L for studies in palliative thoracic radiotherapy because those instruments provide a measure of QOL specific to patients with lung cancer or lung metastases.

Change in urinary markers of osteoclast activity following palliative radiotherapy for bone metastases.

AIMS: To examine the effect of radiotherapy for bone metastases on urinary markers of osteoclast activity. MATERIALS AND METHODS: Patients with radiological evidence of bone metastases planned for palliative radiotherapy were eligible for the study. A urine specimen was collected before and 1 month after radiotherapy to assess levels of calcium, creatinine, magnesium, phosphate, N-telopeptide and pyridinoline. The Brief Pain Inventory was completed in person at baseline and by telephone follow-up at 1 month after radiotherapy. Patients were classified as responders (complete or partial pain response) or non-responders (stable or progressive pain) to radiotherapy based on the International Bone Metastases Consensus Criteria for end point measurements. Absolute values of urine markers were compared between responders and non-responders, or between responders and patients with progression. RESULTS: Our study population consisted of 74 men and 51 women. A single 8 Gy or 20 Gy in five daily fractions were commonly employed. At the 1 month follow-up, all Brief Pain Inventory functional interference scores showed a highly significant decrease from baseline (P<0.01). From our study population, 58 (64%) were classified as responders and 57 (46%) as non-responders to radiotherapy. We compared the urinary markers between the responders and the non-responders. There were no statistically significant differences between the two groups either in terms of baseline markers or in terms of month 1 follow-up markers. There was no significant change from baseline to the 1 month follow-up in responders or in non-responders to radiotherapy. CONCLUSION: Baseline levels of urinary markers could not predict which patient would benefit from palliative radiotherapy.

Demographic profile and utilization statistics of a Canadian inpatient palliative care unit within a tertiary care setting.

BACKGROUND: Canadian data describing inpatient palliative care unit (PCU) utilization are scarce. In the present study, we performed a quality assessment of a 24-bed short-term PCU with a 3-months-or-less life expectancy policy in a tertiary care setting. METHODS: Using a retrospective chart review, we explored wait time (wt) for admission (May 2005 to April 2006), length of stay [los (February 2005 to January 2006)], and patient demographics. RESULTS: The wt data showed 508 referrals, with 242 resulting in admissions (92% malignant diagnoses) and 266 not (82% malignant). The most common malignancies in both groups were gastrointestinal, lung, and genitourinary. Median wt for admitted patients was 6 days, varying with referral source, such as the same hospital, home, or another hospital (6, 4, and 8.5 days respectively). Most admissions (93%) occurred in 21 or fewer days. Patient death (52%), admission to another PCU (25%), and declined offer (10%) were common reasons for no admission. Median los for 219 admitted patients was 19 days (range: 0-249 days). Most patients (94%) died in the PCU; a minority were discharged. CONCLUSIONS: Many patients requiring PCU services are admitted within a few days of referral, especially patients with the least available support: those at home. However, half of the non-admitted patients die while waiting-a potential area for improvement. The los for admitted patients complied with the 3-month "expected lifespan" PCU policy. Results are significant, because ensuring quality of life for palliative care patients includes timely PCU access and sufficient los to address end-of-life needs.

Brain metastasis from an unknown primary, or primary brain tumour? A diagnostic dilemma.

Brain metastasis is increasingly common, affecting 20%-40% of cancer patients. After diagnosis, survival is usually limited to months in these patients. Treatment for brain metastasis includes whole-brain radiation therapy, surgical resection, or both. These treatments aim to slow progression of disease and to improve or maintain neurologic function and quality of life.Although less common, primary brain tumours produce symptoms that are similar to those of brain metastasis. Glioblastoma, the most common malignant tumour of the brain, has a median survival of less than 12 months. Patients are often treated with surgical resection followed by radical radiation therapy and chemotherapy.Here, we present 2 separate cases of lesions in the brain radiologically compatible with brain metastasis. In both cases, no primary cancer site had been established, and neurosurgical intervention was sought to obtain a pathologic diagnosis. Both cases were pathologically confirmed as glioblastoma. These cases demonstrate the importance of differentiation between brain metastases and primary brain tumours to ensure that the appropriate management strategy is implemented.

After radiotherapy, do bone metastases from gastrointestinal cancers show response rates similar to those of bone metastases from other primary cancers?

PURPOSE: Reports investigating whether the response rates to palliative radiation therapy (RT) for painful bone metastases from gastrointestinal (GI) cancers are similar to rates for bone metastases from other primary cancer sites have been limited. The present study evaluated response rates for symptomatic bone metastases from gi cancers after palliative outpatient rt in the Rapid Response Radiotherapy Program (RRRP). PATIENTS AND METHODS: We identified 69 patients with bone metastases from gi primaries who received palliative rt in the RRRP clinic during 1999-2006. We extracted records for 31 of these patients during 1999-2003 from an RRRP database that used the Edmonton Symptom Assessment Scale (ESAS). Record for the remaining 38 patients during 2003-2006 were extracted from an RRRP database that used the Brief Pain Inventory (BPI). Eligibility criteria for encryption in the two RRRP databases and for collection of patient demographic information (age, sex, primary cancer site, and Karnofsky performance status) were identical. Response rates for this cohort of metastatic gi patients were then compared to rates for 479 patients receiving palliative RT for bone metastases from other primary cancer sites. Pain scores from the ESAS and BPI and data on analgesic consumption were collected at baseline and by telephone follow-up at 4, 8, and 12 weeks after RT for all patients. Complete (CR), partial (PR), and overall (CR+PR) responses were evaluated according to International Consensus Endpoints. RESULTS: Assessment of the 69 patients with metastatic GI cancers revealed CR, PR, and CR+PR rates of 18%, 42%, and 61% at 4 weeks; 22%, 35%, and 57% at 8 weeks; and 50%, 21%, and 71% at 12 weeks for evaluable patients. The 479 evaluable patients with metastatic cancer from other primary cancer sites had CR, PR, and CR+PR rates of 25%, 27%, and 51% at 4 weeks; 26%, 22%, and 48% at 8 weeks; and 22%, 29%, and 51% at 12 weeks. No statistically significant differences were observed in RT response rates for bone metastases from GI cancers than from other primary cancer sites. CONCLUSIONS: After palliative RT, bone metastases from gi cancers demonstrate response rates that are similar to rates for metastases from other primary cancer sites. Patients with symptomatic bone metastases from GI malignancies should be referred for palliative RT as readily as patients with osseous metastases from other primary cancer sites.

Radiation recall dermatitis: case report and review of the literature.

"Radiation recall"-also called "radiation recall dermatitis"-has been defined as the "recalling" by skin of previous radiation exposure in response to the administration of certain response-inducing drugs. Although the phenomenon is relatively well known in the medical world, an exact cause has not been documented. Here, we report a rare occurrence of the radiation recall phenomenon in a breast cancer patient after palliative radiotherapy for bone, brain, and orbital metastases.

Did the pattern of practice in the prescription of palliative radiotherapy for the treatment of uncomplicated bone metastases change between 1999 and 2005 at the rapid response radiotherapy program?

AIMS: Since 1999, randomised clinical trials and meta-analyses have reported equal efficacy of pain relief from single- and multiple-fraction radiotherapy for bone metastases. A number of factors, including limited radiotherapy resources, waiting times, and patient convenience, suggest single fraction to be the treatment of choice for patients. However, international patterns of practice indicate that multiple fractions are still commonly used. This study examined whether dose-fractionation schemes used for the treatment of bone metastases at the Rapid Response Radiotherapy Program (RRRP) at the Odette Cancer Centre have changed since 1999. MATERIALS AND METHODS: A retrospective review of the prospective RRRP database and hospital records were conducted for all patients treated with palliative radiotherapy for uncomplicated bone metastases at the RRRP in 1999 (or baseline), 2001, 2004 and from 1 January to 31 July 2005. Data were collected on patient demographics and clinical characteristics. RESULTS: Of the 693 patients, 65 and 35% were prescribed single fraction (predominantly single 8 Gy) and multiple fractions (predominantly 20 Gy/five fractions), respectively. The administration of single treatments generally increased over time, from 51% in 1999 to 66% in 2005 (P=0.0001). On the basis of multiple logistic regression analyses, patients were more likely to be prescribed single-fraction radiotherapy if they had prostate cancer, had a poorer performance status, were treated to the limbs, hips, shoulders, pelvis, ribs, scapula, sternum, or clavicle (compared with the spine), were treated by a radiation oncologist who had been trained in earlier years, and who were treated after 1999. CONCLUSIONS: Between 1999 and 2005, the use of single-fraction radiotherapy increased, corresponding to publications showing equal efficacy of pain relief between single and multiple fractions in the management of uncomplicated bone metastases. However, about a third of patients still received multiple fractions.

Improving community knowledge of obstetric fistula prevention and treatment.

In recent years, information has emerged in the scientific literature regarding the experience of physicians and clinical staff who try to prevent and treat obstetric fistulas at health facilities in developing countries. However, little is known about attempts to match the services they provide with policy and programmatic interventions in the communities they serve. To determine what is known about community involvement in the prevention and treatment of obstetric fistulas, the authors inquired into experiments carried out by EngenderHealth and partners in 15 African and Asian countries. From raw data, gray literature, and personal communications with those working in the field, they learned that engaging the community may be the keystone in the eradication of fistulas in low-resource settings, and they learned how to engage the community.

Percutaneous cementoplasty of lytic metastasis in left acetabulum.

Minimally invasive procedures such as percutaneous cementoplasty can provide immediate pain relief and can restore mechanical stability for patients with bone metastases who are not candidates for surgery or who show resistance to radiotherapy or analgesic treatment. Here, we examine a case of percutaneous cementoplasty to treat a lytic lesion of the acetabulum from breast cancer. Good filling was observed, and no complications occurred. A research assistant recorded the patient's scores on the Karnofsky Performance Scale, Townsend Functional Assessment Scale, and Brief Pain Inventory before surgery and at days 1, 2, and 4 and weeks 1, 2, and 4 post-procedure. Improvement in pain and walking ability was demonstrated within the first 48 hours of treatment, and that improvement remained constant throughout follow-up. These findings echo the literature, in that percutaneous cementoplasty provides immediate and long-term pain relief with few complications. We recommend that percutaneous cementoplasty be used as an additional tool for palliative treatment of patients with bone metastases.

New multidisciplinary prostate bone metastases clinic: first of its kind in Canada.

Prostate cancer is the most common non-skin malignancy in men. Almost all men who die from prostate cancer have hormone-refractory prostate cancer with metastasis to bone. Emerging supportive treatments-including chemotherapy, bisphosphonates, and surgery-require integration that is optimized in a multidisciplinary setting. A multidisciplinary clinic for bone metastases has been in place at Toronto-Sunnybrook Regional Cancer Centre since 1999, combining orthopedic surgery, radiation oncology, interventional radiology, and palliative medicine for all patients with bone metastases. The addition of a prostate-focused multidisciplinary clinic integrates these services for patients with advanced prostate cancer.

An innovative rapid response radiotherapy program to reduce waiting time for palliative radiotherapy.

BACKGROUND: Waiting for radiation therapy (RT) in Ontario has been a major problem for the past decade. In 1996, the Toronto-Sunnybrook Regional Cancer Centre (TSRCC) initiated a Rapid Response Radiotherapy Program (RRRP) to provide timely palliative RT for symptom relief of patients with terminal cancer. PURPOSE: This study reviews the clinical activity of the RRRP over the past 8 years to evaluate if we are meeting the objectives of the program. MATERIALS AND METHODS: From the TSRCC oncology patient information system (OPIS) database, we retrieved the number of patients referred to the RRRP, their demographics, diagnosis and treatment for the calendar years 1996 to 2003 inclusive. We calculated the time interval between referral to consultation, consultation to simulation and the percentage of cases who started RT on their initial consultation for all new cases referred to the RRRP. RESULTS: From 1996 to 2003, the number of cases seen in consultation increased from just over 200 cases annually in the first 2 years to about 500 cases per year in the last 4 years, for a total of 3,290. There were 1,792 (54%) men and 1,498 (46%) women; median age was 69 years (range, 21-95 years). Breast, lung, genitourinary and gastrointestinal primaries accounted for over 80% of all referrals. The majority was referred for palliative treatment of symptomatic bone (70%) or brain (14%) metastases. The dose fractionation for bone metastases was a single 8-Gy fraction in 45%, 20 Gy in five fractions in 42%, 30 Gy in ten fractions in 4% and other dose fractionations in 9%. Nearly 90% were seen within 2 weeks of referral (38% within 1 week). Eighty-five percent were simulated on the day of their initial consultation. Sixty percent started their RT treatment on the day of their consultation visit. The overall median interval from referral to treatment was 8 days. CONCLUSION: Over the past 8 years, the annual number of new cases referred to the RRRP has doubled. The overall median interval from referral to consultation was 8 days. Sixty percent were simulated, planned and started treatment on the day of their initial consultation. We therefore are meeting our goal of providing rapid access to palliative RT for symptomatic cancer patients.

The do-not-resuscitate order: incidence of documentation in the medical records of cancer patients referred for palliative radiotherapy.

Patients with symptomatic metastases referred for outpatient palliative radiotherapy for symptom control at the Rapid Response Radiotherapy Program (RRRP) and the Bone Metastases Clinic (BMC) at the Toronto-Sunnybrook Regional Cancer Centre have a limited life expectancy. Relevant medical information is missing from the files of many referred patients when they arrive at the clinics, potentially causing delayed treatment and ambiguity in the best management of their needs in situations of worsening condition. Clear documentation of the do-not-resuscitate (DNR) order is imperative to avoid panic and the taking of unnecessarily aggressive measures in situations in which cardiopulmonary resuscitation (CPR) has no benefit or is not desired. Here, we report the current practices of CPR code status documentation for patients referred to the RRRP and the BMC for out-patient palliative radiotherapy.We reviewed referral notes and accompanying medical records for 209 consecutive patients seen in the RRRP and the bmc during May-August 2004 for documentation of CPR-related advance directives. Patient demographics and cancer history were also recorded.Only 13 (6.2%) of the 209 patients had any documented reference to CPR code status. Of these 13 patients, 8 were DNR-coded, and 5 were full code. As compared with patients having no documented cpr code status, patients with documented status were significantly older (median age: 77 years; p = 0.0347), had poorer performance status (median Karnofsky performance status score: 40; p = 0.0001), and were more likely to be referred hospital inpatients (69%, p = 0.0004).Only a small proportion of symptomatic advanced cancer patients had any documentation of CPR code status upon referral for outpatient palliative radiotherapy. In future, our clinics plan to request information about CPR code status on our referral form.

Solitary bone metastasis beneath the shoulder shield: coincidence or cause?

Post-mastectomy radiotherapy has been demonstrated to improve locoregional control in breast cancer patients. We report a case involving a 44-year-old breast cancer patient who presented with a solitary bone metastasis in the area beneath the shoulder shield, likely from a coincidental recurrence.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.