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Considering a rejection rate of 80–90%, the preparation of a research grant is often considered a daunting task since it is resource intensive and there is no guarantee of success, even for seasoned researchers. This commentary provides a summary of the key points a researcher needs to consider when writing a research grant proposal, outlining: (1) how to conceptualise the research idea; (2) how to find the right funding call; (3) the importance of planning; (4) how to write; (5) what to write, and (6) key questions for reflection during preparation. It attempts to explain the difficulties associated with finding calls in clinical pharmacy and advanced pharmacy practice, and how to overcome them. The commentary aims to assist all pharmacy practice and health services research colleagues new to the grant application process, as well as experienced researchers striving to improve their grant review scores. The guidance in this paper is part of ESCP’s commitment to stimulate “innovative and high-quality research in all areas of clinical pharmacy”.

While there is a lot of documented evidence about the clinical and cost effectiveness of pharmacists' role extensions there is an inherent gap between service development and implementation.This study aims to better understand the complex factors that influence the implementation of clinical pharmacy services from both the perspective of the community pharmacy service providers and service developers.A prospective qualitative interview study using purposive sampling of twelve service developers and twelve community pharmacy service providers from across all nine Federal States of Austria. The validated and piloted interview guide contained questions and prompts on role perceptions, attitudes, experience, implementation barriers, training needs and measures identified to strengthen clinical pharmacy provision in community pharmacy. Verbatim quotes were independently mapped to the Framework for the Implementation of Services in Pharmacy (FISpH) by two researchers.24 Interviews were carried out. Data saturation was achieved. There is a great deal of enthusiasm to develop the remit of clinical pharmacy services. It is seen as important to ensure the future survival of the profession. Service developers are more positive and confident in the implementation success and pharmacists' skills than providers. Clear mandates for politics, academia and individual pharmacists have been discussed to affect change.Austrian pharmacists are facing the same well documented challenges as many other healthcare systems only with more urgency. The development of a clinical pharmacy service framework; education accreditation standard and a well-supported continuous professional development system are considered key to bring about the necessary culture shift.

Objective: To explore the patients’ experiences on initiation of treatment with antineoplastic medicines for colorectal cancer., Design: A qualitative study using semistructured, face-to-face interviews based on the adapted patients’ lived experience with medicines conceptual model was conducted. Ethics approval was granted. Interviews took place while eligible patients were receiving their first cycle of antineoplastic medicines, audiorecorded and transcribed verbatim. Interpretative phenomenological analysis was used., Settin: National oncology hospital in Malta., Participants: Adult patients over 18 years, newly diagnosed with colorectal cancer and initiating treatment with 5-fluorouracil/folinic acid and oxaliplatin (FOLFOX) or capecitabine plus oxaliplatin (XELOX) between October 2018 and September 2019 were recruited. Patients were included if fully aware of their diagnosis and the treatment being received. A purposive sample of 16 patients participated in the study., Results: Five themes emerged from our analysis: (1) beliefs and knowledge on cancer and treatment; (2) healthcare system in relation to treatment; (3) medicine-taking practice; (4) medicine-related impact and (5) personal support structure. In addition, 16 subthemes were identified to describe the interplay of all aspects of the patients’ experiences on initiation of treatment with antineoplastic medicines., Conclusion: This qualitative study illustrated that the patients find themselves in a complex situation on initiation of treatment with antineoplastic medicines. The patients’ knowledge on their specific treatment and active participation in making an informed decision need to be addressed. The healthcare system should develop tailored patient-centred services that specifically address the lack of patient empowerment and patient’s concern about treatment specific information., peer-reviewed

BackgroundGeneral practice in the UK is experiencing a crisis. Greater multidisciplinary working is a potential solution. The new general practice contract in Scotland encourages this and includes a new pharmacotherapy service to be delivered by General Practice Clinical Pharmacists (GPCPs). Consensus is lacking for the standards of practice for delivery of pharmacotherapy medication reviews (which are polypharmacy and chronic medication reviews) as part of this service.AimTo identify and validate standards of practice for polypharmacy and chronic disease medication (pharmacotherapy level 3) reviews conducted by GPCPs. MethodA two-phased mixed-methods consensus methodology was used. Phase 1: An expert group of GPCPs (n=4) and clinical pharmacist managers (n=2) responsible for delivering the pharmacotherapy service used a Modified Nominal Group Technique to generate potential standards. Phase 2: Two-round Delphi survey involving GPCPs with ≥1 year of experience of working in general practice (n=159).ResultsThe expert group identified 44 potential standards of practice for polypharmacy and chronic disease reviews. Practicing GPCPs indicated during the Delphi phase that the 44 standards were applicable to practice. The standards of practice covered seven main categories: skills, environment, qualifications, qualities and behaviours, knowledge, process and experience.ConclusionPracticing GPCPs indicated that the standards identified by the expert group are acceptable and valid for current practice and the delivery of polypharmacy and chronic medication reviews. The application of these standards to practice may help GPCPs and general practices to ensure equitable delivery of patient care.

Background General practice in the UK is experiencing a crisis. Greater multidisciplinary working is a potential solution. The new general practice contract in Scotland encourages this and includes a new pharmacotherapy service to be delivered by General Practice Clinical Pharmacists (GPCPs). Consensus is lacking for the standards of practice for delivery of pharmacotherapy medication reviews (which are polypharmacy and chronic medication reviews) as part of this service. Aim To identify and validate standards of practice for polypharmacy and chronic disease medication (pharmacotherapy level 3) reviews conducted by GPCPs. Method A two-phased mixed-methods consensus methodology was used. Phase 1: An expert group of GPCPs (n = 4) and clinical pharmacist managers (n = 2) responsible for delivering the pharmacotherapy service used a Modified Nominal Group Technique to generate potential standards. Phase 2: Two-round Delphi survey involving GPCPs with ≥ 1 year of experience of working in general practice (n = 159). Results The expert group identified 44 potential standards of practice for polypharmacy and chronic disease reviews. Practicing GPCPs indicated during the Delphi phase that the 44 standards were applicable to practice. The standards of practice covered seven main categories: skills, environment, qualifications, qualities and behaviours, knowledge, process and experience. Conclusion Practicing GPCPs indicated that the standards identified by the expert group are acceptable and valid for current practice and the delivery of polypharmacy and chronic medication reviews. The application of these standards to practice may help GPCPs and general practices to ensure equitable delivery of patient care.

Introduction Cancer is a complex illness whereby patients require access to various healthcare services provided by the multidisciplinary team over a prolonged period of time. Aim To explore patients’ experiences of cancer healthcare services whilst undergoing treatment with antineoplastic medicines. Methods This qualitative study involved in-depth semi-structured interviews with 16 patients diagnosed with colorectal cancer at the only national oncology centre available in Malta. Interviews allowed a detailed exploration of the patients’ lived experience with antineoplastic treatment on aspects relating to medicine-related beliefs, practice and burden (1). Interviews were held between December 2018 and November 2019, when patients were receiving their fourth or sixth cycle of treatment with XELOX or FOLFOX respectively. They were audio-recorded and transcribed verbatim in the native language, with a sample of 10% checked for transcription accuracy. Data saturation was established using a stopping criterion of three (2). Data were analysed thematically by two independent researchers with disagreements resolved through discussion. Results A total of 16 patients with colorectal cancer, aged between 38 and 78 years, were interviewed. Four themes emerged:1) experiences of patients of the cancer services, 2) healthcare professionals’ communication with patients, 3) patient’s perception of the role of the healthcare professional in the management of the illness, and 4) recommendations for the service. Patients were appreciative of the fast-track route for hospital appointments upon a diagnosis of cancer and the accessibility of a cancer-specific nurse navigator to address their challenges. Problems were mainly encountered in-between cycles while patients were at home, with difficulty in assessing the severity of adverse effects. This led to struggles with decision making and whether to self-refer to the emergency department or alternatively whether to seek verbal advice from the nurse navigator. Despite the generalised information provision during the nurse-led group information session at initiation of treatment, patients recommended an additional follow-up session for reinforcement of knowledge tailored to their own needs. Although most patients preferred to receive parenteral treatment under the surveillance of trained healthcare professionals in a hospital setting, they proposed activities such as individual or group counselling and educational treatment-specific sessions or peer-led discussion during treatment administration. ‘Initially I had no symptoms however these cropped up around 4th cycle. Having meetings with healthcare professionals whilst receiving chemo allows sharing of information about this specific treatment.’[P004] Recommendations made by patients included medicine review taking into account their chronic medicines and provision of knowledge to assess the severity of their adverse effects experienced at home ‘Due to my heart problems, I wonder whether another ‘safe’ medicine may be used…prefer to discuss this with healthcare professional whilst having a review of all my medicines.’[P003] Conclusion This study highlighted that timeliness and accessibility of healthcare services are crucial in cancer care and are greatly appreciated by the patients. The limitation of the study was that views were only captured from those participants who consented to be interviewed. The introduction of interventions along the treatment journey to address their current needs was recommended. References (1) Brincat A, Bonanno PV, Stewart D, Weidmann AE. Patients’ lived experiences with antineoplastic medicines for the management of malignant solid tumours: A systematic review. Research in Social and Administrative Pharmacy. 2020; 16(4):450-463. (2) Francis JJ, Johnston M, Robertson C, Glidewell L, Entwistle V, Eccles MP et al. What is an adequate sample size? Operationalising data saturation for theory-based interview studies. Psychol Health. 2010; 25(10):1229–1245.

The digital skeleton of the hindlimbs was evaluated radiographically in 27 standing Brown Swiss heifers. The lateral digital skeleton was significantly longer than its medial counterpart on both a hard (wooden block) and a softer (wooden block covered with a thin rubber mat) surface. There were no significant differences between lengths measured on the hard and the soft surface. The difference between the length of the lateral and medial digits originated at the level of the condyles and increased with P1 and P2. It was partially compensated by a higher third phalanx (P3) in the medial digit, but this did not offset the overall length difference. The findings of this study confirmed that the length asymmetry of the paired digits of cattle documented previously in post-mortem specimens is also present in living cattle. Further investigation is required to determine the clinical relevance of the length asymmetry of the digital skeleton in cattle.

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumours and is still associated with a poor prognosis in advanced disease. To improve the standard therapy with gemcitabine, we initiated a prospective randomised phase-II trial with gemcitabine (GEM) versus gemcitabine plus sunitinib (SUNGEM) based on data of in vitro trials and phase-I data for the combination treatment. The rational of adding sunitinib was its putative antiangiogenic mechanism of action. Methods A total of 106 eligible patients with locally advanced, unresectable or metastatic PDAC without previous system therapy were randomised to receive GEM at a dosage of 1.000 mg/m 2 d1, 8, 15 q28 versus a combination of SUNGEM at a dosage of GEM 1.000 mg/m 2 d1 + 8 and sunitinib 50 mg p.o. d1–14, q21d. The primary end-point was progression free survival (PFS), secondary end-points were overall survival (OS), toxicity and overall response rate (ORR). Results The confirmatory analysis of PFS was based on the intend-to-treat (ITT) population ( N = 106). The median PFS was 13.3 weeks (95% confidence interval (95%-CI): 10.4–18.1 weeks) for GEM and 11.6 weeks for SUNGEM (95%-CI: 7.0–18.0 weeks; p = 0.78 one-sided log-rank). The ORR was 6.1% (95%-CI: 0.7–20.2%) for GEM and for 7.1% (95%-CI: 0.9–23.5%) for SUNGEM ( p = 0.87). The median time to progression (TTP) was 14.0 weeks (95%-CI: 12.4–22.3 weeks) for GEM and 18.0 weeks (95%-CI: 11.3–19.3 weeks) for SUNGEM ( p = 0.60; two-sided log-rank). The median OS was 36.7 weeks (95%-CI: 20.6–49.0 weeks) for the GEM arm and 30.4 weeks (95%-CI: 18.1–37.6 weeks) for the SUNGEM ( p = 0.78, one-sided log-rank). In regard to toxicities, suspected SAEs were reported in 53.7% in the GEM arm and 71.2% in the SUNGEM arm. Grade 3 and 4 neutropenia was statistically significantly higher in the SUNGEM arm with 48.1% versus 27.8% in the GEM arm ( p = 0.045, two sided log-rank). Conclusions The combination SUNGEM was not sufficient superior in locally advanced or metastatic PDAC compared to GEM alone in regard to efficacy but was associated with more toxicity.

Summary Background Tofacitinib is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis. The Oral Rheumatoid Arthritis triaL (ORAL) Strategy aimed to assess the comparative efficacy of tofacitinib monotherapy, tofacitinib plus methotrexate, and adalimumab plus methotrexate for the treatment of rheumatoid arthritis in patients with a previous inadequate response to methotrexate. Methods ORAL Strategy was a 1 year, double-blind, phase 3b/4, head-to-head, non-inferiority, randomised controlled trial in patients aged 18 years or older with active rheumatoid arthritis despite methotrexate therapy. Patients were randomly assigned (1:1:1) to receive oral tofacitinib (5 mg twice daily) monotherapy, oral tofacitinib (5 mg twice daily) plus methotrexate, or subcutaneous adalimumab (40 mg every other week) plus methotrexate at 194 centres in 25 countries. Eligible patients received live zoster vaccine at investigators' discretion. The primary endpoint was the proportion of patients who attained an American College of Rheumatology response of at least 50% (ACR50) at month 6 in the full analysis set (patients who were randomly assigned to a group and received at least one dose of the study treatment). Non-inferiority between groups was shown if the lower bound of the 98·34% CI of the difference between comparators was larger than −13·0%. This trial is registered with ClinicalTrials.gov, number NCT02187055. Findings 1146 patients received treatment (384 had tofacitinib monotherapy; 376 had tofacitinib and methotrexate; and 386 had adalimumab and methotrexate). At 6 months, ACR50 response was attained in 147 (38%) of 384 patients with tofacitinib monotherapy, 173 (46%) of 376 patients with tofacitinib and methotrexate, and 169 (44%) of 386 patients with adalimumab and methotrexate. Non-inferiority was declared for tofacitinib and methotrexate versus adalimumab and methotrexate (difference 2% [98·34% CI −6 to 11]) but not for tofacitinib monotherapy versus either adalimumab and methotrexate (−6 [−14 to 3]) or tofacitinib and methotrexate (−8 [−16 to 1]). In total, 23 (6%) of 384 patients receiving tofacitinib monotherapy, 26 (7%) of 376 patients receiving tofacitinib plus methotrexate, and 36 (9%) of 386 patients receiving adalimumab plus methotrexate discontinued due to adverse events. Two (1%) of the 384 patients receiving tofacitinib monotherapy died. No new or unexpected safety issues were reported for either treatment in this study for up to 1 year. Interpretation Tofacitinib and methotrexate combination therapy was non-inferior to adalimumab and methotrexate combination therapy in the treatment of rheumatoid arthritis in patients with an inadequate response to methotrexate in this trial. Tofacitinib monotherapy was not shown to be non-inferior to either combination. Funding Pfizer Inc.

ZusammenfassungChronisch-traumatische Schädigungen des Sprunggelenks gehören zu den häufigsten Technopathien an den Gliedmaßen beim Rind. Die Prävalenz liegt heutzutage in vielen Betrieben zwischen 40 und 70%, was im Hinblick auf das Wohlergehen der Kühe bedenklich ist. Diese Schädigungen stellen vor allem Indikatoren für die unzureichende Tiergerechtheit der Liegeboxen und der Liegeflächen dar. Haarverlust und Dekubitalstellen an Sprunggelenken sind zudem mit Lahmheiten und weiteren Gliedmaßenkrankheiten, aber auch Eutererkrankungen und verringerter Milchleistung assoziiert. Deshalb haben sie nicht nur für das Wohlbefinden, sondern auch für die Leistung von Milchkühen Bedeutung. Epidemiologische Studien und Verhaltensuntersuchungen zeigen, dass – korrekte Abmessungen der Liegeboxen vorausgesetzt – unter den verschiedenen Liegeflächen die Stroh-Mist-Matratze sowie Sand hinsichtlich der Prävalenz von Liegeschäden am besten abschneiden.

SummaryA two-year-old Braunvieh heifer was presented with a traumatic luxation of the second phalanx of the medial digit and concurrent subluxation of the second phalanx of the lateral digit of the right hindlimb. Closed reduction of both luxations was possible. Surgical arthrodesis was achieved using one narrow 4.5 mm three-hole equine locking compression plate for each joint. Placement of the bone plates resulted in stable arthrodesis of both proximal interphalangeal joints of the right hindlimb but there was persistent residual lameness. The heifer delivered a healthy calf but was slaughtered eight months after surgery because of varus deformity of the contralateral limb. Radio-graphs taken post-mortem revealed pronounced periosteal reactions involving both proximal interphalangeal joints and only partial bony bridging of the joint spaces.

Valproic acid has been demonstrated to mediate cytotoxic effects against tumor cells by acting as a histone-deacetylase inhibitor. However, to date, there are only limited data on the effects of valproic acid in colon cancer. Moreover, information regarding combinations of the drug with chemotherapeutic agents is very limited. The latter is of interest as there is increasing evidence for synergism between so-called "molecular targeting drugs" and chemotherapy. We first demonstrated that valproic acid dose-dependently reduced the viability of adenocarcimona cell lines. After co-incubation with a variety of chemotherapeutic agents, only valproic acid in combination with mitomycin C consistently induced synergistic growth inhibition in all cell lines. To confirm these results in an ex vivo situation, five samples of fresh colon cancer cells were studied. Again, the effect of valproic acid on the viability of the fresh tumor cells was dose dependent. In four of five samples of freshly isolated colon cancer cells, the synergistic effect of valproic acid and mitomycin C on the inhibition of cell growth was confirmed by calculation of the combination index by multiple drug effect analysis. In conclusion, this is the first demonstration that valproic acid as a model substance for histone-deacetylase inhibitors is effective in tumor cells freshly isolated from patients with colon cancer and that the combination of mitomycin C and valproic acid synergistically decreases viability of colon cancer cells.

Background. The reliability of the rapid urease test has not been proven in patients with peptic ulcer bleeding. Some studies show bad diagnostic results with the rapid urease test for gastrointestinal bleeding. Aims. To evaluate the efficacy of the rapid urease test in patients with bleeding gastric or duodenal ulcers. Patients and methods. A total of 96 patients with acute peptic ulcer bleeding without proton pump inhibitor or antibiotic therapy within the last 14 days before bleeding were included into the study. During index endoscopy, specimens for histological and rapid urease test were obtained from the antrum and corpus mucosa of the stomach. Patients were also investigated by the 13C-urea breath test. Diagnostic quality parameters were calculated with the histology and the 13C-urea breath test as reference and compared with a matched control group with uncomplicated ulcers. Results. The sensitivity of the rapid urease test was 80% and the specificity 100% compared to histology and 13C-urea breath test. The negative predictive value was 75%. These values were statistically significantly different from those of the control group (sensitivity 96%, specificity 100%, negative predictive value 88%). Conclusion. The exclusive use of the rapid urease test cannot be recommended in patients with peptic ulcer bleeding.

The claws of pastured Scottish Highland Cattle are large and this may raise the question if regular claw trimming is necessary. Therefore, the claws of the right thoracic and pelvic limbs were measured in 22 Scottish Highland cows 4 times 8 weeks apart. The cows were kept on various alpine pastures before the first measurement, on a two-hectare low-land pasture before the second measurement, in a welfare-compliant straw-bedded free stall before the third measurement and on alpine pasture before the fourth measurement. Housing conditions significantly affected claw dimensions. The claws were composed of dry, hard horn during pasture periods, and had prominent weight-bearing hoof-wall borders and soles with a natural axial slope. Long dorsal walls and heels and a greater symmetry were common. Claw lesions were absent. In contrast, free-stall housing was associated with shorter toes and steeper toe angles, but white line deterioration, heel horn erosion, wearing of the axial slope and hoof wall edges were common.Die Klauen von extensiv gehaltenen schottischen Hochland-Rindern scheinen oft zu gross und werfen die Frage nach der Notwendigkeit einer regelmässigen Klauenpflege auf. Bei 22 schottischen Hochland-Kühen wurden daher die Klauen der rechten Schulter- und Beckengliedmassen an 4 Terminen im Abstand von je 8 Wochen vermessen. Vor dem ersten Messtermin wurden die Kühe auf der Alp, vor dem zweiten auf einer ca. 2 ha grossen Talweide, vor dem dritten in einem Tiefstreu-Offenstall mit betonierter Lauffläche und vor dem vierten wieder auf einer Alp gehalten. Die Klauenmasse änderten sich signifikant mit der Haltungsumgebung. Nach Weideperioden waren zwar lange Dorsalwände und lange Ballen, aber auch eine grössere Symmetrie zwischen den Klauen vorhanden. Die relativ grossen Klauen wiesen ein hartes, trockenes Horn, überstehende Tragränder und eine natürliche Hohlkehlung auf. Anzeichen für Klauenerkrankungen waren nicht sichtbar. Nach Laufstallhaltung waren die Dorsalwände kürzer und die Winkel steiler, doch waren Schädigungen wie angegriffene weisse Linien, Ballenhornfäule, fehlende Hohlkehlung und abgelaufene Trageränder die Regel.Les onglons des vaches Higland détenues de façon extensive semblent souvent trop grands et posent la question de la nécessité de soins réguliers. On a donc mesuré 4 fois à intervalle de 8 semaines les onglons des membres antérieur et postérieur droits de 22 Higland. Les vaches étaient détenues avant la première mesure à l'alpage, avant la deuxième sur un pâturage de plaine d'environ 2 ha, avant la troisième dans une stabulation libre ouverte à litière profonde avec des surfaces de sortie bétonnées, et avant la quatrième à nouveau à l'alpage. Le volume des onglons se modifiait significativement selon les conditions de détention. Après les périodes de pâturage, on observait de longues parois dorsales et de longs talons mais aussi une grande symétrie entre les onglons. Les onglons relativement grands présentaient une corne dure et sèche, des parois débordantes et une concavité naturelle. Il n'y avait pas de signe de pathologie des onglons. Après la détention en stabulation, les parois dorsales étaient plus courtes et leur angle plus redressé, mais on constatait régulièrement des dégâts comme une ligne blanche altérée, de la pourriture au niveau des talons, un manque de concavité ou des parois usées.Spesso, gli unghioni dei bovini di razza Highlander scozzese, nelle forme estensive di detenzione, sembrano troppo grandi e fanno riflettere sulla necessità di una cura regolare. In 22 mucche di razza Highlander scozzese sono stati misurati 4 volte a intervalli di 8 settimane l'una dall'altra, gli unghioni della spalla destra e degli arti posteriori. La prima volta, i bovini erano ancora tenuti sull'alpeggio, la seconda su un pascolo di ca. 2 ettari a valle, la terza in stalla aperta a lettiera profonda con un'area di movimento cementata e la quarta di nuovo sull'alpe. Le dimensioni degli unghioni variavano chiaramente in relazione al luogo di detenzione. Dopo i periodi di messa al pascolo si sono riscontrate muraglie e talloni molto allungati e una grande simmetria tra gli unghioni. Questi grandi unghioni presentavano un corno molto duro e secco, margini sporgenti e una scanalatura naturale. Segni di malattie dell'unghione non erano visibili. Dopo la stabulazione libera, le muraglie si erano accorciate e l'angolo era più marcato tuttavia si riscontravano regolarmente danni quali: linee bianche rovinate, erosio ungulae, scanalature mancanti e margini consumati.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumours and is still associated with a poor prognosis in advanced disease. To improve the standard therapy with gemcitabine, we initiated a prospective randomised phase-II trial with gemcitabine (GEM) versus gemcitabine plus sunitinib (SUNGEM) based on data of in vitro trials and phase-I data for the combination treatment. The rational of adding sunitinib was its putative antiangiogenic mechanism of action.A total of 106 eligible patients with locally advanced, unresectable or metastatic PDAC without previous system therapy were randomised to receive GEM at a dosage of 1.000mg/m(2) d1, 8, 15 q28 versus a combination of SUNGEM at a dosage of GEM 1.000mg/m(2) d1+8 and sunitinib 50mg p.o. d1-14, q21d. The primary end-point was progression free survival (PFS), secondary end-points were overall survival (OS), toxicity and overall response rate (ORR).The confirmatory analysis of PFS was based on the intend-to-treat (ITT) population (N=106). The median PFS was 13.3 weeks (95% confidence interval (95%-CI): 10.4-18.1 weeks) for GEM and 11.6 weeks for SUNGEM (95%-CI: 7.0-18.0 weeks; p=0.78 one-sided log-rank). The ORR was 6.1% (95%-CI: 0.7-20.2%) for GEM and for 7.1% (95%-CI: 0.9-23.5%) for SUNGEM (p=0.87). The median time to progression (TTP) was 14.0 weeks (95%-CI: 12.4-22.3 weeks) for GEM and 18.0 weeks (95%-CI: 11.3-19.3 weeks) for SUNGEM (p=0.60; two-sided log-rank). The median OS was 36.7 weeks (95%-CI: 20.6-49.0 weeks) for the GEM arm and 30.4 weeks (95%-CI: 18.1-37.6 weeks) for the SUNGEM (p=0.78, one-sided log-rank). In regard to toxicities, suspected SAEs were reported in 53.7% in the GEM arm and 71.2% in the SUNGEM arm. Grade 3 and 4 neutropenia was statistically significantly higher in the SUNGEM arm with 48.1% versus 27.8% in the GEM arm (p=0.045, two sided log-rank).The combination SUNGEM was not sufficient superior in locally advanced or metastatic PDAC compared to GEM alone in regard to efficacy but was associated with more toxicity.

The claws of pastured Scottish Highland Cattle are large and this may raise the question if regular claw trimming is necessary. Therefore, the claws of the right thoracic and pelvic limbs were measured in 22 Scottish Highland cows 4 times 8 weeks apart. The cows were kept on various alpine pastures before the first measurement, on a two-hectare low-land pasture before the second measurement, in a welfare-compliant straw-bedded free stall before the third measurement and on alpine pasture before the fourth measurement. Housing conditions significantly affected claw dimensions. The claws were composed of dry, hard horn during pasture periods, and had prominent weight-bearing hoof-wall borders and soles with a natural axial slope. Long dorsal walls and heels and a greater symmetry were common. Claw lesions were absent. In contrast, free-stall housing was associated with shorter toes and steeper toe angles, but white line deterioration, heel horn erosion, wearing of the axial slope and hoof wall edges were common.

Background and objective Because of the paucity of information on the epidemiology of acute poisoning requiring intensive medical care, all such patients treated on the medical intensive care unit of the university hospital in Frankfurt am Main, Germany, between January 1993 and December 1999, were retrospectively evaluated. Patients and methods Of the total of 6211 patients, 147 (80 women, 67 men, mean age 41 years, 2,3 %) were treated for acute intoxication in the intensive care unit. Results Reasons for admission to the intensive care unit were the need for ventilator treatment or intensive monitoring of vital functions. 52 % of the patients (n = 76) had attempted suicide, most of them using anti-depressive drugs (n = 19), paracetamol (n = 16), or benzodiazepines (n = 9). Two patients (2,6 %) died. 48 % of the patients (n = 71) were admitted because of accidental poisoning. Leading toxic agents in this group were heroin (n = 19), alcohol (n = 18) and digitalis (n = 12). 11 patients had taken herbicides, animal poisons or chemicals used at work or for house cleaning. In this cohort, three i. v. drug abusers (4,2 %) had died. Depending on the agents used, a variety of treatments (charcoal, antidots, extracorporal therapy) were undertaken. Conclusion Due to excellent care in the prehospital phase and in the emergency room the number of patients requiring treatment on the intensive care unit was rather low. The mortality was in the range of other reports.

Prostate apoptosis response gene-4, known as par-4, is a new proapoptotic factor functionally required but not sufficient for apoptosis. Since there is evidence from prostate cancer cells that par-4 is involved in regulation of bcl-2 we assessed expression of par-4 and bcl-2 in different populations of normal and neoplastic lymphocytes.Expression of par-4 mRNA and protein in different subpopulations of normal and neoplastic lymphocytes was assessed by reverse transcription polymerase chain reaction and Western blot.Par-4 mRNA was not detectable in lymphocytes of healthy volunteers (n = 10), but was present in the majority of samples of chronic lymphocytic leukemia (n = 30), chronic lymphocytic leukemia/prolymphocytic leukemia (n = 6) and acute lymphocytic leukemia (n = 10). Par-4 protein was expressed unanimously in samples of mononuclear cells from healthy volunteers and patients with CLL, but less frequently in immature lymphocytes, including neoplastic cells of CLL/PLL and ALL. The decreased frequency of par-4 expression in immature subpopulations was confirmed by results on lymphocytic cell lines at various stages of maturation. Comparing the expressional patterns of par-4 and bcl-2 there was an inverse relationship of both proteins in ALL and different lymphocytic cell lines, indicating a functional relationship of par-4 and bcl-2.This study establishes par-4 as a factor expressed in the majority of normal and neoplastic lymphocytic cells, demonstrating a decreased frequency of protein expression in less differentiated lymphocytes and an inverse expressional pattern of par-4 and bcl-2 in lymphocytic cell lines and ALL.

Hock lesions are the most common husbandry-related disease of the locomotor system in dairy cattle. These conditions are referred to as technopathies. The prevalence of tarsal lesions ranges from 40 to 70% and thus, from a welfare standpoint, clearly exceeds an acceptable level. These lesions usually indicate inadequate stall or cubicle design in dairy barns. The presence and severity of hock lesions are associated with lameness and other limb disorders as well as mastitis and reduced milk yield. Hock lesions therefore influence animal welfare and production. Epidemiological and behavioural studies have shown that a manure pack covered with straw is associated with the lowest prevalence of lesions, followed by sand bedding, provided that the lying space is adequate.

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2-75)) were given 240 μg/kg plerixafor SC 9-11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 10(6) CD34(+) cells/μL. The median cell yield was 3.35 × 10(6) CD34+ cells/kg (0-29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 10(6) CD34+ cells/kg (1.6-5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 10(6) CD34+ cells/kg (0-24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

Unter dem Begriff T-Zell-Lymphom und/oder T-Zell-Leukamie ist eine sehr heterogene Gruppe von Neoplasien der T-Zell-Reihe zusammengefast, die sich in diversen Klassifikationen wiederfinden. Von Bedeutung ist, insbesondere wegen des Verlaufs und der Therapie, die Einteilung in unreife (lymphoblastische) und sog. periphere Lymphome/Leukamien. Wahrend die unreifen T-Zell-Neoplasien von T-Zell-Prakursorzellen aus dem Knochenmark und dem Thymus abstammen, gehen periphere T-Zell-Neoplasien aus T-Zellen hervor, die die thymische Differenzierung durchlaufen haben (peripher des Thymus). Histologische Details sind unter der Darstellung der einzelnen Entitaten beschrieben.

The development of inhibitors to factor VIII may cause life-threatening bleedings, and presents a serious clinical management problem. Acquired hemophilia is a rare event, with an incidence of 0.2–1 per million per year and a mortality between 6% and 22%. In the majority of patients no cause can be identified. Up to 50% of the patients with autoantibodies against factor VIII are associated with autoimmune diseases, malignancies, drugs, pregnancies, or the postpartum period. The aims of management in acquired hemophilia are to eradicate the FVIII autoantibodies and to treat the acute bleeding episodes.

The reliability of the rapid urease test has not been proven in patients with peptic ulcer bleeding. Some studies show bad diagnostic results with the rapid urease test for gastrointestinal bleeding.To evaluate the efficacy of the rapid urease test in patients with bleeding gastric or duodenal ulcers.A total of 96 patients with acute peptic ulcer bleeding without proton pump inhibitor or antibiotic therapy within the last 14 days before bleeding were included into the study. During index endoscopy, specimens for histological and rapid urease test were obtained from the antrum and corpus mucosa of the stomach. Patients were also investigated by the 13C-urea breath test. Diagnostic quality parameters were calculated with the histology and the 13C-urea breath test as reference and compared with a matched control group with uncomplicated ulcers.The sensitivity of the rapid urease test was 80% and the specificity 100% compared to histology and 13C-urea breath test. The negative predictive value was 75%. These values were statistically significantly different from those of the control group (sensitivity 96%, specificity 100%, negative predictive value 88%).The exclusive use of the rapid urease test cannot be recommended in patients with peptic ulcer bleeding.

Background and study aims Eradication of Helicobacter pylori infection can reduce the rebleeding rate of peptic ulcer bleeding in the long term. There are few data on the influence of H. pylori on the rebleeding rate in the acute phase of bleeding however. We therefore prospectively investigated the influence of H. pylori infection on the early rebleeding rate in patients who had undergone successful endoscopic hemostasis treatment for peptic ulcer bleeding. Patients and methods Between January 1996 and November 2000 all patients with peptic ulcer bleeding were evaluated consecutively. The diagnosis of H. pylori infection was made at index endoscopy, using histology and the rapid urease test. Bleeding activity was assessed using the Forrest classification. After successful endoscopic hemostasis all patients received omeprazole 40 mg or pantoprazole 40 mg, intravenously, twice a day for 3 days. Rebleeding episodes were recorded over 21 days following primary hemostasis. Results 344 patients were enrolled into the study. The prevalence of H. pylori infection was 62.9 %. A total of 51 patients showed rebleeding (14.8 %), of whom 31 were H. pylori-positive (60 %). There was no statistically significant difference between the H. pylori-positive and -negative patients, however. The rebleeding rate did not differ between patients with H. pylori infection alone and patients also using nonsteroidal anti-inflammatory drugs. When stratifying patients according to activity of bleeding at index endoscopy, we were also unable to find any significant influence of H. pylori infection on the outcome of Forrest class I and IIa bleedings. Conclusion Based on our data, it can be concluded that H. pylori infection does not affect the early rebleeding rate in patients with peptic ulcer bleeding after successful endoscopic hemostasis.

Because of the paucity of information on the epidemiology of acute poisoning requiring intensive medical care, all such patients treated on the medical intensive care unit of the university hospital in Frankfurt am Main, Germany, between January 1993 and December 1999, were retrospectively evaluated.Of the total of 6211 patients, 147 (80 women, 67 men, mean age 41 years, 2,3 %) were treated for acute intoxication in the intensive care unit.Reasons for admission to the intensive care unit were the need for ventilator treatment or intensive monitoring of vital functions. 52 % of the patients (n = 76) had attempted suicide, most of them using anti-depressive drugs (n = 19), paracetamol (n = 16), or benzodiazepines (n = 9). Two patients (2,6 %) died. 48 % of the patients (n = 71) were admitted because of accidental poisoning. Leading toxic agents in this group were heroin (n = 19), alcohol (n = 18) and digitalis (n = 12). 11 patients had taken herbicides, animal poisons or chemicals used at work or for house cleaning. In this cohort, three i. v. drug abusers (4,2 %) had died. Depending on the agents used, a variety of treatments (charcoal, antidots, extracorporal therapy) were undertaken.Due to excellent care in the prehospital phase and in the emergency room the number of patients requiring treatment on the intensive care unit was rather low. The mortality was in the range of other reports.

Regulation of apoptotic cell death is being increasingly recognized as a mechanisms by which cytostatic agents mediate tumor cell death. Preliminary clinical studies with bendamustine, an alkylating agent with a purine nucleus, provide strong evidence that this drug is a highly effective cytostatic in low grade lymphomas. Therefore, we investigated the in vitro activity of bendamustine in combination with other established cytotoxic drugs.2 lines (DOHH-2, WSU-NHL) and mononuclear cells (MNC) from patients with leukemic low-grade B-non-Hodgkin's lymphoma (NHL) (n=10), T-NHL (n=7) and chronic lymphocytic leukemia (CLL) (n=12). Apoptosis (7-AAD), depolarization of mitochondrial membrane potential (MMP, JC-1), caspase-3-activity (FIENA) and cell proliferation (XTT/WST-1) were determined. Several incubation times and drug dosages (for IC(30/50/75/90)) were studied. Synergistic, additive or antagonistic effects were calculated by a median plot effect and the combination index (CI) method.In general, combinations of bendamustine with mitoxantrone or doxorubicin resulted in antagonistic effects in the tested cell lines and the MNC from the patients. CI-calculation failed in these cases since there was not a sufficient dose response. On the other hand, the combination of bendamustine with 2-CdA showed synergistic in vitro activity on the tested cell lines, neoplastic lymphocytes from patients with peripheral T-cell lymphomas and partially on MNC from patients with CLL and B-NHL. The antagonism of the combination of bendamustine and anthracyclines appeared to be due to inhibition of depolarization of mitochondrial-membrane potential and caspase-3-activity during apoptosis of the studied cell lines.In conclusion, our results suggest that schedules using combinations of bendamustine and anthracyclines should not be recommended for the treatment of low-grade NHL, whereas bendamustine combined with 2-CdA could be considered for the development of future treatment strategies.

We describe a case of cytotoxic T-large granular lymphocyte leukaemia showing typical morphological features, expressing antigens characteristic for cytotoxic T cells and exhibiting marked natural killer-like cytotoxicity towards different target cells. Moreover, characterization of the T-cell receptors revealed simultaneous expression of two different types of beta-chains as well as alpha-chains by the malignant cell clone. The patient had an 8 year history of indolent disease, before progressing to an aggressive clinical course hardly responsive to chemotherapeutic treatment. This is the first description of a peripheral T-cell neoplasm expressing four distinct types of T-cell receptor molecules.

Background In patients with Iron Deficiency Anemia (IDA) occult gastrointestinal bleeding is generally investigated by bidirectional endoscopy. The aim of our study was to examine the long-term follow-up of patients with IDA where the sources of bleeding couldn't be detected despite close endoscopic and radiologic examination of the GI tract. Methods Based on the endoscopic data base we examined consecutive patients who were referred for gastrointestinal endoscopy due to IDA with a negative endoscopic (upper GI endoscopy and colonoscopy) evaluation. Further diagnostic work up (repeated endoscopy of the upper and lower GI tract by an experienced investigator, small bowel enteroclysis, push enteroscopy, proctoscopy, intraoperative enteroscopy, angiography, scintigraphic examinations) was recorded. The eligible patients were divided into 2 groups: Group 1 (no identification of the source of bleeding in the GI tract); group 2 (source of gastrointestinal blood loss was found). Long-term follow-up was performed by telephone interview with patients and/or with their general practitioner. Results 79 patients (mean age 58.8 years [17-83, 44] female) with IDA met the inclusion criteria. In 42 patients (53%) the endoscopic and radiographic evaluation was unable to find the source of gastrointestinal blood loss. 29 of these patients (69%) showed a resolved anemia after a mean follow-up of 48 months (18 months-5 years). 10 patients had a mild anemia, 3 required blood transfusions. In group one Helicobacter pylori infection was significantly more prevalent in comparison with group 2 (57% vs. 38%, p = 0.032). Conclusion Based on our data, the prognosis of IDA with negative endoscopy is favorable. The pathogenic role of Helicobacter pylori infection should be evaluated in further studies.

Hepatosplenic gd T-cell lymphoma is a rare entity of peripheral T-cell lymphomas. We characterized in detail the first case of hepatosplenic gd -T-cell lymphoma following acute myeloid leukemia.Hepatosplenic gd -T-cell lymphoma was diagnosed in a woman who had been in complete remission (CR) of acute myeloid leukemia (AML) for two years. Improvement but no objective response of the disease was observed after various types of chemotherapy. CR was achieved after related donor stem cell transplantation. Thirteen months later relapse of hepatosplenic gd T-cell lymphoma was diagnosed. While being prepared for a second transplantation the patient developed meningeal lymphoma and died. The patient's lymphoma cells were studied by immunologic, functional and molecular techniques.Lymphoma cells expressed the gd T-cell receptor (TCR), CD2, CD3, CD5, CD7, CD38, CD45, CD161 (NKR-P1), TIA and Ki67. Further analysis revealed expression of Vd1 and two distinct TCRg chains, Vg3 and Vg9, by the malignant cell clone. The clonality of the T-cells was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) followed by sequencing of TCR Vg3, Vg9 and Vd1 junctional regions. Clone-specific PCR was negative at diagnosis of AML and was positive at all times during follow-up of the hepatosplenic gd T-cell lymphoma. The lymphoma cells mediated strong natural killer cell-like cytotoxic activity, possibly explained by expression of CD161 and a lack of killer inhibitory receptor.Several so far undescribed features were observed in this case of hepatosplenic gd T-cell lymphoma, such as T-cell lymphoma following AML, expression of two distinct T-cell receptor g-chains, and an unexpected cytotoxic phenotype.

The current report describes the experience from the Frankfurt AIDS Cohort Study with patients suffering from renal failure. The clinical data of 4993 HIV-infected patients between 1983 and 1998 were analyzed retrospectively. Patients were seen at least twice a year and clinical features, routine laboratory results, including CD4+ cell counts, concomittant diseases, and antiretroviral therapy were documented by standardized methods. The incidence of renal failure during 4 observation periods with different antiretroviral treatment strategies are compared and data are discussed. Within the 16 years of observation 47 patients with impairement of their kidney function were identified. A trend to an increase of RF could be documented (chi superset2 -for trend p = 0.0246). The additional review intends to summarize the diverse reasons leading to renal dysfunction in HIV-infected individuals with special emphasis on glomerular disease and renal complications related to HIV therapy.

A 60-year-old woman developed a periorbital lipogranuloma after endonasal surgery on her paranasal sinuses. The granulomatous inflammation was caused by nonabsorbable lipids introduced by the postoperative nasal tamponade which was soaked in antibiotic ointment. These lipids were transported into the periorbital tissue by the postoperative hemorrhage. Since the course of the inflammatory process is chronic, surgical removal is the best treatment.

Recent reports suggest that complex interactions exist between the neuroendocrine and immune systems. It has been shown for example that cytokines are able to stimulate the hypothalamo-pituitary-adrenal axis. In addition, some studies present evidence that endothelin is able to modulate the activity of several hypothalamic-pituitary axes, e.g. by inducing the ACTH production.We investigated the effects of interleukin-2 on endothelin levels and the hypothalamo-pituitary-adrenal axis. We determined the interleukin-6, big-endothelin, endothelin-1, ACTH, cortisol and AVP responses to intravenously and subcutaneously administered interleukin-2 in 8 cancer patients in a randomized placebo controlled trial.8 Patients (2 female and 6 male), age 44 +/- 4.8 years, were enrolled. All patients had a World Health Organization performance status of 1 or less and a Karnofsky Index of at least 80%.Blood-samples were taken before and 15, 30, 45, 60, 120, 180, 240, 300 and 360 min after interleukin-2 injection. Cytokine serum levels and the plasma levels of big-endothelin, endothelin, ACTH and AVP were analysed using radioimmuno-assays. Cortisol was assayed by an enzyme-linked immunosorbent assay.Interleukin-2 treatment significantly increased plasma big-endothelin levels (P0.01 vs basal) and endothelin-1 levels (P0.05 vs basal) within two hours and this was followed by an increase in ACTH (P0.01 vs basal) and cortisol (P0.05 vs basal) within three hours. Interleukin-6 levels increased two hours after interleukin-2 administration (P0.01 vs basal). Interleukin-2 had no detectable effect on AVP, blood pressure or heart rate.Our data demonstrate that cytokines are able to activate the human hypothalamo-pituitary-adrenal axis in vivo. On the basis of the observed time kinetics and in connection with previous findings from in vitro and animal models, we conclude that endothelin may be a link between cytokines and corticotrophin-releasing hormone, most probably functioning as a cytokine-induced neuromodulator controlling pituitary functions.

The Wilms' tumor gene (wt1) is strongly expressed in malignant blasts of acute myeloid leukemia (AML) in approximately 80% of all cases. However, the role of wt1 expression in non malignant hematopoietic cells remains unclear. To characterize the expression of wt1 in differentiating hematopoietic progenitors, we isolated and cultured CD34+ progenitor cells from four healthy bone marrow donors with stem cell factor (SCF) and granulocyte colony stimulating-factor (G-CSF) to induce differentiation into granulocytes. Four different cultures were carried out for 12 days. During culture, wt1 mRNA expression was analyzed by defining its ratio relative to beta-actin using reverse transcriptase polymerase chain reaction (RT-PCR). To monitor the stage of differentiation, expression of cell surface markers and peroxidase was analyzed daily. The initial purity of CD34+ cells ranged between 80% and 90%; after 12 days, the frequency of neutrophil bands and segmented neutrophils was approximately 60%. Using RT-PCR to determine the ratio of wt1 to beta-actin expression, we reproducibly detected maximum expression of wt1 mRNA at day 0 in two cultures and at day 1 in two other CD34+ cell cultures; at both these time points nearly all cells fulfilled the morphological and immunephenotypical criteria of early hematopoietic blast cells. Wt1 expression dropped rapidly at day 1 and 2, respectively, in these two pairs of cultures, and was accompanied by an increase of cells expressing CD33 surface antigen. Our data suggest that wt1 expression is restricted to a subset of CD34+ progenitors and downregulated in later stages of differentiation in vitro.

We report on studies of multiple ionization and fragmentation of free Hgn (n ≤ 80) clusters in the femtosecond time domain at wavelengths ranging from 255 nm to 800 nm. After excitation by single laser pulses of an intensity of 5 * 1011 W/cm2 we observe prompt formation of multiply charged Hgn clusters. The Hgn cluster size distribution observed up to n ≈ 80 shows in additon to singly charged also doubly and triply charged clusters with a surprisingly high amount of doubly charged clusters. The measured cluster size distribution is nearly independent of laser wavelengths. For higher laser intensities (2 * 1012 W/cm2) we observe multiply charged mercury atoms up to Hg5+. At 1013 W/cm2 molecules and clusters eventually disappear due to Coulomb explosion and complete Fragmentation. Only atomic ions, singly and multiply charged, with high kinetic energies are then observed.

In acute myeloblastic leukemia (AML), the T cell response and cytotoxic activity are impaired at time of diagnosis due to not-yet-identified soluble immunosuppressing factors. The inhibition of autologous antileukemic immune response by these factors may support immunosurveillance of AML. A well-known inhibitor of lymphokine-activated killer (LAK) cell activity is transforming growth factor-beta 1 (TGF-beta 1). To evaluate the possible significance of TGF-beta 1 for the impaired cytotoxic activity in AML at time of diagnosis, we looked for the TGF-beta 1-specific mRNA, for the production and release of TGF-beta 1, and for its relevance for immunosuppressing activities in AML. In the culture supernatants of 18 investigated AMLs, we detected various amounts of TGF-beta protein. The TGF-beta 1 and TGF-beta 2 protein concentrations were 105 pg/mL (50-240 pg/mL) and 32 pg/mL (2-91 pg/mL), respectively. In 13 of 15 patients, the leukemic blasts expressed TGF-beta 1 mRNA. To exclude possible interferences with contaminating mononuclear cells (MNC), the data were confirmed by analysis of sorted blast cells and leukemic cell lines. All investigated leukemic cell lines expressed TGF-beta 1 protein and mRNA. The culture supernatants of AMLs inhibited LAK activity strongly in a dose-dependent manner. The inhibition of cytotoxicity could be restored by the addition of neutralizing TGF-beta 1 antibodies. The data suggest TGF-beta 1 to be a relevant factor for the inhibition of cytotoxic activities in AMLs.

The Wilms' tumor gene (wt-1) is expressed in the developing fetal kidney, gonads and in Wilms' tumors. Recently, the expression of wt-1 in leukemia-derived cell lines and cases of acute leukemias (AL) was reported. The present study was designed to investigate the potential of wt-1 as genetic marker for acute myelocytic leukemias (AML). Blast cells from 52 patients with AML, 14 patients in complete remission (CR) and four leukemic cell lines were examined for expression of wt-1 mRNA. Peripheral blood mononuclear cells (PBMNC) and bone marrow (BM) from 13 healthy persons were used as negative controls. RNA of the wt-1 gene was expressed in 41/52 (79%) patients with previously untreated AML. The majority of the 14 patients studied in CR lost wt-1 expression. In three out of the four patients in CR reappearance of wt-1 expression preceded relapse of the disease. In three of the four tested cell lines wt-1 specific transcription was demonstrated. No correlation to FAB classification, immunophenotype or response to treatment was found. Our experiments indicate wt-1 expression in the majority of AML, but not in bone marrow or PBMNC of healthy controls. Therefore, wt-1 expression may be associated with the presence of malignant blast cells and the analysis of wt-1 gene expression via PCR may be a sensitive method for the detection of leukemic blast cells.

Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) of a patient with von Hippel-Lindau disease and renal cell carcinoma were studied for the T-cell receptor beta chain variable region (TCR-V beta) repertoire. The patient was vaccinated with irradiated autologous tumor cells from a renal tumor mass, a vaccine-draining lymph node was removed, and lymphocytes were cultured in the presence of autologous tumor cells and low-dose interleukin 2 (IL2). These lymphocytes were adoptively transferred to the patient together with systemic IL2 (30,000 IU/kg every 8 h). Analysis of TCR-V beta expression was performed by polymerase chain reaction in PBL before, during, and after therapy, in vaccine-draining lymph node lymphocytes, and in TIL obtained from moderately infiltrated, nonresponding renal tumor mass and from a more intensely infiltrated lung metastasis, which was responding to treatment. Significant differences in the expression of TCR-V beta 13.1 by T-cells recovered from these various sites were observed. Also, TIL recovered from the responding lung metastasis and cultured in the presence of IL2 gave rise to autologous tumor-reactive CD4+ T-cells, whereas the nonresponsive renal tumor yielded a mixture of T- and natural killer cells. In PBL obtained prior to treatment and during IL2 therapy, expression of V beta 13.1 was 0.7 and 1.8%, respectively, of the total V beta gene repertoire. Fresh vaccine-draining lymph node lymphocytes contained 5.9% of V beta 13.1-expressing T-cells. After IL2 therapy, V beta 13.1 gene expression increased to 5.4% in PBL. In the nonresponding tumor mass, the frequency of V beta 13.1 gene expression among TIL was 12%, whereas in the responding, highly infiltrated nodule, it was 28%, with a striking loss of expression of other V beta gene families. Sequencing of the amplified product of V beta 13.1 complementary DNA from the responding pulmonary metastasis showed restrictions in the complementarity-determining region 3. Thus, in vivo expansion of V beta 13.1-expressing CD4+ T-cells, possibly in response to a tumor-associated antigen, occurred in the responding tumor mass following this form of therapy and correlated with tumor course.