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This study was carried out in Sardinia, an Italian region with a very high IDDM incidence. HLA class I and class II antigens were studied in 97 unrelated IDDM patients, 33 complete families with at least one affected member each, and 559 healthy controls. Molecular typing of the DQB1 alleles was carried out in 31 patients and 61 controls. The haplotypes were determined by family studies. The HLA-DR3, DQw2, and DR4 antigens were positively associated with IDDM. The DR3 antigen was nearly always associated to B18 and frequently carried by the extended haplotype A30 Cw5 B18 3F130 DR3 DQw2. The genotype analysis of the patients showed a strong increase of the DR3/DR4 heterozygotes with a relative risk higher than that of the DR3 and DR4 homozygotes. The DR2 antigen was negatively associated with IDDM in the central island districts but not in the southern districts. The DQB1 molecular analysis showed only three alleles in the patients: DQB1*0201 (75.8 per cent), DQB1*0302 (16.1 per cent), and DQB1*0502 (8.1 per cent). These alleles are non Asp 57, so it would seem that nearly if not all Sardinian IDDM patients are NA/NA homozygotes. The DQB1*0502 allele, extremely rare in other Caucasian populations, represents in Sardinia about 70 per cent of the HLA-DR2 haplotypes, contributing to the increase of the pool of IDDM susceptible genes. Moreover it is carried in 27 per cent of the DR2 positive individuals with the extended haplotype A2 Cw7 Bw58 3F31 DR2 DQw1.AZH.

A 25-year-old woman with AIDS was submitted to HLA-identical allogeneic BMT after cytoablation with busulphan and cyclophosphamide and combined anti-HIV-1 therapy with zidovudine, IFN-alpha 2 and anti-HIV-1-specific T cell clones. Marrow engraftment occurred after 18 days and tests for HIV-1 were negative after 30 days but the hematologic reconstitution of the patient was poor. A second BM infusion from the same donor was ineffective and treatment with GM-CSF only induced a transient increase of the blood cell count, suggesting iatrogenic damage to the BM microenvironment. The development of ARDS led to the death of the patient 10 months after transplantation. Post-mortem investigation did not reveal any active infections and PCR on autopsy tissues was negative for HIV-1.

We report a case of BMT from an unrelated donor (MUD) in a patient affected by Wiskott- Aldrich syndrome (WAS). The donor-recipient pair was completely identical for two HLA-extended haplotypes. The conditioning regimen consisted of Bu 14 mg/kg followed by CY 200 mg/kg. GVHD prophylaxis was carried out with CsA plus short-term MTX. Allogeneic engraftment was obtained without any signs of acute or chronic GVHD. At fifteen months from the transplant the patient was in an excellent clinical condition. This case confirms the role of BMT from MUD in WAS, and suggests that complete donor-recipient identity for two entire HLA-extended haplotypes is a particularly favorable immunogenetical condition in this type of transplant.

A 16-year-old Sardinian girl affected by homozygous beta-thalassaemia was submitted to allogeneic BMT using an HLA-identical, MLC-negative, unrelated donor. The donor and the patient were homozygous for the entire extended haplotype A30, Cw5, B18, F130, DRB1*0301, DRB3*0202, DQA1*0501, DQB1*0201 and heterozygous for DPB1*0301/DPB1*0202. The conditioning regimen consisted of 14 mg/kg busulphan and 160 mg/kg cyclophosphamide. Engraftment was achieved 14 days from BMT and the haematological reconstitution was complete without any signs of acute or chronic GVHD. Seven months after the transplant the patient was in excellent general condition. The hypothesis is advanced that when two HLA extended haplotypes are shared by donors and recipients, particularly in homozygosity, this is a very favourable immunogenetic condition in unrelated BMT.

An impaired intestinal epithelial barrier is thought to be a major factor in the pathogenesis of human inflammatory bowel disease (IBD). IBD is frequently investigated by inducing a damaged barrier in murine models of colitis. This can be done by feeding mice with dextran sulfate sodium (DSS) polymers in their drinking water. Refinement measures should focus on alleviating unnecessary suffering during this probably painful condition. Appropriate parameters are needed to decide when to terminate the experiments. Our aim was to investigate whether a change in burrowing behaviour is a sensitive measure of animal welfare in murine models of colitis. Acute colitis was induced in C57BL/6 mice with 2.0% DSS over nine days. The burrowing test is based on the species-typical behaviour of mice to spontaneously displace items from tubes within their home cage. As a burrowing apparatus, a water bottle (250 mL, 150 mm length, 55 mm diameter) filled with 138–142 g of pellets of the animal’s diet was used. The presence of intestinal inflammation as a result of acute DSS-induced colitis was confirmed by a decrease in body weight, colon length and an increase of murine endoscopic index of colitis severity, histological score and spleen weight in the group receiving DSS as compared with the control group. An onset of intestinal inflammation correlated with a significant decrease in burrowing behaviour ( P

This prospective study compared diagnostic and prognostic value of conventional cytologic (CC) examination and flow cytometry (FCM) of baseline samples of cerebrospinal fluid (CSF) in 174 patients with newly diagnosed aggressive non-Hodgkin lymphoma (NHL). FCM detected a neoplastic population in the CSF of 18 of 174 patients (10%), CC only in 7 (4%; P < .001); 11 patients (14%) were discordant (FCM+/CC−). At a median follow-up of 46 months, there were 64 systemic progressions and 10 CNS relapses, including 2 patients with both systemic and CNS relapses. Two-year progression-free and overall survival were significantly higher in patients with FCM− CSF (62% and 72%) compared with those FCM+ CSF (39% and 50%, respectively), with a 2-year CNS relapse cumulative incidence of 3% (95% confidence interval [CI], 0-7) versus 17% (95% CI, 0-34; P = .004), respectively. The risk of CNS progression was significantly higher in FMC+/CC− versus FCM−/CC− patients (hazard ratio = 8.16, 95% CI, 1.45-46). In conclusion, FCM positivity in the CSF of patients with high-risk NHL is associated with a significantly higher CNS relapse risk and poorer outcome. The combination of IV drugs with a higher CNS bioavailability and intrathecal chemotherapy is advisable to prevent CNS relapses in FCM+ patients.

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas. In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2. Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5. Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

Detection of persistent pain of a mild-to-moderate degree in laboratory mice is difficult because mice do not show unambiguous symptoms of pain or suffering using standard methods of short-term observational or clinical monitoring. This study investigated the potential use of burrowing performance — a spontaneous and highly motivated behavior — as a measure of post-operative pain in laboratory mice. The influence of minor surgery on burrowing was investigated in adult C57BL/6J mice of both genders in a modified rodent burrowing test (displacement of food pellets from a pellet-filled tube) within the animal’s home cage. Almost all (98%) healthy mice burrowed (mean latency 1.3 h, SEM 0.5 h). After surgery without pain treatment, latency of burrowing was significantly prolonged (mean ∆ latency 10 h). Analgesic treatment using the anti-inflammatory drug carprofen (5 mg/kg bodyweight) decreased latency of burrowing after surgery (mean ∆ latency 5.5 h) to the level found in mice that had been anaesthetised (mean ∆ latency 5.3 h) or had received anaesthesia and analgesia (mean ∆ latency 4.6 h). Analgesia during surgery was associated with a significantly earlier onset of burrowing compared to surgery without pain treatment. A distinct gradation in burrowing performance was found ranging from the undisturbed pre-operative status to the intermediate level following anaesthesia/analgesia and surgery with analgesia, to the pronounced prolongation of latency to burrow after surgery without pain relief. In conclusion, post-surgical impairment of general condition, probably mainly attributable to pain, can be conveniently assessed in laboratory mice on the basis of the burrowing test.

Isoflurane is currently the most common volatile anaesthetic used in laboratory mice, whereas in human medicine the more modern sevoflurane is often used for inhalation anaesthesia. This study aimed to characterize and compare the clinical properties of both anaesthetics for inhalation anaesthesia in mice. In an approach mirroring routine laboratory conditions (spontaneous breathing, gas supply via nose mask, preventing hypothermia by a warming mat) a 50 min anaesthesia was performed. Anaesthetics were administered in oxygen as carrier gas at standardized dosages of 1.5 minimum alveolar concentrations, which was 2.8% for isoflurane and 4.9% for sevoflurane. Both induction and recovery from anaesthesia proceeded quickly, within 1–2 min. During anaesthesia, all reflex testing was negative and no serious impairment of vital functions was found; all animals survived. The most prominent side-effect during anaesthesia was respiratory depression with hypercapnia, acidosis and a marked decrease in respiration rate. Under anaesthesia, heart rate and core body temperature remained within the normal range, but were significantly increased for 12 h after anaesthesia. Locomotor activity, daily food and water consumption and body weight progression showed no abnormalities after anaesthesia. No significant difference was found between the two anaesthetics. In conclusion, isoflurane and sevoflurane provided an equally reliable anaesthesia in laboratory mice.

Background The laboratory mouse is commonly used as a sophisticated model in biomedical research. However, experiments requiring major surgery frequently lead to serious postoperative complications and death, particularly if genetically modified mice with anatomical and physiological abnormalities undergo extensive interventions such as transmitter implantation. Telemetric transmitters are used to study cardiovascular physiology and diseases. Telemetry yields reliable and accurate measurement of blood pressure in the free-roaming, unanaesthetized and unstressed mouse, but data recording is hampered substantially if measurements are made in an exercising mouse. Thus, we aimed to optimize transmitter implantation to improve telemetric signal recording in exercising mice as well as to establish a postoperative care regimen that promotes convalescence and survival of mice after major surgery in general. Results We report an optimized telemetric transmitter implantation technique (fixation of the transmitter body on the back of the mouse with stainless steel wires) for subsequent measurement of arterial blood pressure during maximal exercise on a treadmill. This technique was used on normal (wildtype) mice and on transgenic mice with anatomical and physiological abnormalities due to constitutive overexpression of recombinant human erythropoietin. To promote convalescence of the animals after surgery, we established a regimen for postoperative intensive care: pain treatment (flunixine 5 mg/kg bodyweight, subcutaneously, twice per day) and fluid therapy (600 μl, subcutaneously, twice per day) were administrated for 7 days. In addition, warmth and free access to high energy liquid in a drinking bottle were provided for 14 days following transmitter implantation. This regimen led to a substantial decrease in overall morbidity and mortality. The refined postoperative care and surgical technique were particularly successful in genetically modified mice with severely compromised physiological capacities. Conclusion Recovery and survival rates of mice after major surgery were significantly improved by careful management of postoperative intensive care regimens including key supportive measures such as pain relief, administration of fluids, and warmth. Furthermore, fixation of the blood pressure transmitter provided constant reliable telemetric recordings in exercising mice.

Housing mice in the laboratory in groups enables social interaction and is the way a laboratory should house mice. However, adult males show reciprocal aggression and are therefore frequently housed individually. Alternatively, a grid divider, which allows sensory contact by sight and smell but prevents fighting and injuries, can separate mice within 1 cage. This study examined the influence of this housing method on various physiological and behavioral parameters. Adult male mice housed for 10 days with sensory contact to an unfamiliar male displayed significant increases in heart rate (HR), body core temperature (BT), and motor activity (ACT). Furthermore, the mice suffered impaired nest-building behavior and significantly reduced body weight. Conversely, males housed in a similar manner with a female companion showed only a transient elevation of ACT, BT, and HR. Although no clear beneficial effect of housing males with sensory contact to females was evident, this study could not exclude it. On the other hand, housing of mature males in this way leads to sustained detrimental alterations of physiology and behavior, thus implying severe impairment of animal well-being.

Background Pain of mild to moderate grade is difficult to detect in laboratory mice because mice are prey animals that attempt to elude predators or man by hiding signs of weakness, injury or pain. In this study, we investigated the use of telemetry to identify indicators of mild-to-moderate post-laparotomy pain. Results Adult mice were subjected to laparotomy, either combined with pain treatment (carprofen or flunixin, 5 mg/kg s/c bid, for 1 day) or without pain relief. Controls received anesthesia and analgesics or vehicle only. Telemetrically measured locomotor activity was undisturbed in all animals, thus confirming that any pain experienced was of the intended mild level. No symptoms of pain were registered in any of the groups by scoring the animals' outer appearance or spontaneous and provoked behavior. In contrast, the group receiving no analgesic treatment after laparotomy demonstrated significant changes in telemetry electrocardiogram recordings: increased heart rate and decreased heart rate variability parameters pointed to sympathetic activation and pain lasting for 24 hours. In addition, core body temperature was elevated. Body weight and food intake were reduced for 3 and 2 days, respectively. Moreover, unstructured cage territory and destroyed nests appeared for 1–2 days in an increased number of animals in this group only. In controls these parameters were not affected. Conclusion In conclusion, real-time telemetric recordings of heart rate and heart rate variability were indicative of mild-to-moderate post-laparotomy pain and could define its duration in our mouse model. This level of pain cannot easily be detected by direct observation.