Naïve Pine Terpene Response to the Mountain Pine Beetle (Dendroctonus ponderosae) through the Seasons.

Insect herbivores must contend with constitutive and induced plant defenses. The mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae, Scolytinae) has expanded its range east of the Rocky Mountains into the western boreal forest and is encountering evolutionarily naïve lodgepole pines (Pinus contorta) and jack pines (Pinus banksiana). Pinus contorta and P. banksiana in the expanded range have different constitutive and induced defenses in response to wounding and inoculation with fungal associates of D. ponderosae. In the historic range, previous studies have examined phloem terpene content prior to and just after D. ponderosae mass attack, but the terpene profile of attacked trees post-overwintering is unknown. We examined the response of mature P. contorta and P. banksiana trees to experimentally-applied mass attack by D. ponderosae and quantified phloem terpenes at three time points, pre-attack, post-attack (same season), and the following spring, post-overwintering. Phloem content of total terpenes as well as many individual terpenes increased after D. ponderosae attack but were only significantly higher than pre-attack levels at the post-overwintering time point in both P. contorta and P. banksiana. The absence of a significant increase in phloem terpenes in the month following attack in naïve pines is a potential cause for increased D. ponderosae offspring production reported in naïve P. contorta. Beetle attack density did not influence the phloem terpene profiles of either species and there was no significant interaction between attack density and sampling time on terpene content. High phloem terpenes in trees that are attacked at low densities could prime these trees for defense against attacks in the following season but it could also make these trees more apparent to early-foraging beetles and facilitate efficient mass attack at low D. ponderosae population densities in the expanded range.

Local, Geographical, and Contextual Variation in the Aggregation Pheromone Blend of the Spruce Beetle, Dendroctonus rufipennis (Coleoptera: Curculionidae).

Prior research from trapping experiments using synthetic pheromone components suggested the existence of local and geographical variation in the pheromone system of the spruce beetle, Dendroctonus rufipennis (Kirby). To test hypotheses concerning variation in the spruce beetle's aggregation pheromone blend, we extracted volatiles from the hindguts of individual spruce beetles sampled from sites in eastern Canada (Nova Scotia and Newfoundland) and western Canada (British Columbia and Alberta) and quantified the amounts of four known aggregation pheromone components within each sample. Chiral analyses were performed on a subset of samples. Frontalin (1,5-dimethyl-6,8-dioxabicyclo[3.2.1]octane) was more dominant in western Canada, whereas MCOL (1-methyl-2-cyclohexen-1-ol) and seudenol (3-methyl-2-cyclohexen-1-ol) were more dominant in the east. Verbenene (4-methylene-6,6-dimethylbicyclo[3.1.1]hept-2-ene) was prevalent only in one of our six sites (Rocky Mountain House, AB). Female beetles in Rocky Mountain House (AB) also produced a higher proportion of (+)-frontalin than females from the other sites, and a comparison to previously published data suggests additional geographic variation that was not captured among our sites. When paired with a male in a gallery, female spruce beetles produced pheromone blends with more frontalin and less MCOL compared to solitary female beetles. Our results show that variation in the spruce beetle's pheromone blend exists at both large and small spatial scales, possibly a consequence of local selective pressures and assortative mating.

Use of fertility treatments in BRCA1/2 mutation carriers and risk for ovarian and breast cancer: a systematic review.

PURPOSE: Mutations in the genes BRCA1 and BRCA2 represent a significant risk factor for ovarian and breast cancer. With increasing number and success rates, fertility protection and treatment are gaining importance also for BRCA1/2 mutation carriers. However, the effect on primary cancer risk and risk for recurrence remains unclear. This review analyses the published data on fertility treatment and risk of ovarian and breast cancer in BRCA1/2 mutation carriers. METHODS: In this review, we included all relevant articles published in English from 1995 to 2018. Literature was identified through a search on PubMed and Cochrane Library. RESULTS: We identified one retrospective cohort and one case-control study regarding the association of fertility treatments and ovarian cancer risk in BRCA mutation carriers. The studies show no increase in ovarian cancer risk. Furthermore, one case-control study on the association between fertility treatment and breast cancer risk in BRCA mutation carriers and one prospective cohort study on the long-term safety of medication used for fertility preservation in women with a history of breast cancer were identified. One of the studies shows a possible adverse effect for gonadotropin-containing medication. CONCLUSION: Possible increases in cancer risk associated with fertility treatments in BRCA1/2 mutation carriers cannot be excluded at this time. Based on the existing studies, BRCA1/2 mutation carriers should not be generally excluded from fertility treatments. However, they have to be informed about limited data and possible increases in cancer risk.

Use of oral contraceptives in BRCA mutation carriers and risk for ovarian and breast cancer: a systematic review.

PURPOSE: BRCA mutation carriers have an increased risk of developing breast or ovarian cancer. Oral contraception (OC) is known to increase breast cancer and reduce ovarian cancer risk in the general population. This review analyses the published data on OC and risk of cancer in BRCA mutation carriers. METHODS: We included all relevant articles published in English from 1995 to 2018. Literature was identified through a search on PubMed and Cochrane Library. RESULTS: We included four meta-analyses, one review, one case-control study and one retrospective cohort study on the association between ovarian cancer and OC in BRCA mutation carriers. All report a risk reduction for the OC users and several also describe an inverse correlation with duration of use. Regarding breast cancer, we included four meta-analyses, one review, one case-control study, two case-only studies, one prospective and one retrospective cohort study. Some studies report a risk elevation, while others did not find an association between OC use and breast cancer in BRCA mutation carriers. In other studies, the association was limited to early-onset breast cancer and/or associated with young age at first start of OC. CONCLUSION: Oral contraception leads to a risk reduction of ovarian cancer also in BRCA mutation carriers. An increase in breast cancer risk due to OC cannot be excluded. Women with BRCA mutation who consider OC use have to be informed about possible increase in breast cancer risk and alternative contraceptive methods. OC should not be used for the prevention of ovarian cancer in this population.

Multiple dynamic representations in the motor cortex during sensorimotor learning.

The mechanisms linking sensation and action during learning are poorly understood. Layer 2/3 neurons in the motor cortex might participate in sensorimotor integration and learning; they receive input from sensory cortex and excite deep layer neurons, which control movement. Here we imaged activity in the same set of layer 2/3 neurons in the motor cortex over weeks, while mice learned to detect objects with their whiskers and report detection with licking. Spatially intermingled neurons represented sensory (touch) and motor behaviours (whisker movements and licking). With learning, the population-level representation of task-related licking strengthened. In trained mice, population-level representations were redundant and stable, despite dynamism of single-neuron representations. The activity of a subpopulation of neurons was consistent with touch driving licking behaviour. Our results suggest that ensembles of motor cortex neurons couple sensory input to multiple, related motor programs during learning.

Arterioportal shunting, splanchnic capillary perfusion, and the effects of colloids during capnoperitoneum in neonatal and adolescent pigs.

BACKGROUND: Clinical and experimental data indicate that neonates are sensitive to the CO2 pneumoperitoneum. An impaired splanchnic perfusion during laparoscopy in adults has been reported. We recently confirmed that intravenous colloids improve macrocirculatory function in neonates. We aimed to determine the impact of CO2 pneumoperitoneum on the perfusion of splanchnic organs in the young including effects of colloid application. METHODS: Male piglets (n = 25) were divided into four groups: (1) neonatal controls, (2) neonates with crystalloid restitution, (3) neonates with colloidal restitution, and (4) adolescents with crystalloid restitution. Animals were ventilated and subjected to a 3-h, 10 mmHg CO2 pneumoperitoneum followed by 2 h resuscitation. Hepatic, splanchnic, and arteriovenous shunt perfusion was assessed via central and portal venous catheters. Capillary organ flow was detected by fluorescent microspheres. The rate of bile flow was measured. RESULTS: The neonatal crystalloid group showed a significant decrease in the intestinal capillary perfusion at the end of the recovery period. This was not detectable in the adolescent and colloid group. There was a significant increase in microcirculatory arterioportal shunt flow during the CO2 pneumoperitoneum in both neonatal groups but not in the sham and adolescent groups (p < 0.05). Hepatic arterial perfusion increased after insufflation in all groups and dropped during capnoperitoneum to levels of about 70% baseline. There was no significant impairment of splanchnic perfusion or bile flow as a result of the pneumoperitoneum in all groups. CONCLUSIONS: Capillary perfusion of the abdominal organs was stable during capnoperitoneum and recovery in adolescents and neonates with colloid restitution, but not with crystalloid restitution. Significant arterioportal shunting during capnoperitoneum could affect hepatic microcirculation in neonates. Our data confirm that moderate pressure capnoperitoneum has no major effect on the perfusion of abdominal organs in neonates with adequate substitution.

[Potential of allogeneic bone grafts as antibiotic carriers : Effect of different preparation processes on efficacy]. / Allogene spongiöse Knochenpräparate als Antibiotikaträger : Einfluss verschiedener Aufbereitungsverfahren auf die Wirksamkeit.

BACKGROUND: The rising number of primary joint replacements worldwide is causing an increase of endoprosthetic revision surgery due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome, and the use of antibiotic-impregnated bone grafts might control the infection and provide a good support for the implant. In this study, we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. METHODS: Heat-treated, lyophilized and fresh frozen cryopreserved bone chips were impregnated with gentamicin sulphate, gentamicin palmitate and vancomycin, and calcium carbonate/calcium sulphate treated with antibiotics. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. RESULTS: The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during the entire experiment. This might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high on the first and second days, then decreased and stayed at a low rate until the end of the second week. CONCLUSION: Depending on the surgical strategy, either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within 2 weeks after surgery. In the case of uncemented revision of joint replacements, allogeneic bone can deliver therapeutic doses of gentamicin and peak levels immediately and a fortnight after implantation.

Micro fluorescence in situ hybridization (µFISH) for spatially multiplexed analysis of a cell monolayer.

We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term µFISH. This µFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes - the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed µFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports.

Susceptibility of Lenticels within the Stem Depression of Tomato Fruit to Bacterial Soft Rot.

The postharvest development of bacterial soft rot in tomato fruit caused by Pectobacterium carotovorum is herein linked with inoculation of lenticel-like apertures located around the stem attachment. Stem scars misted with aqueous cell suspensions of P. carotovorum (1 × 106 CFU/ml) or briefly (5 s) immersed in the suspension were likely to become infected during subsequent storage, with a disease incidence exceeding 70% within 7 days at 24°C. Water soaking was initially observed beneath the fruit surface at the juncture of radial walls of endocarp tissues and a corky ring around the stem attachment. If fruit were swirled for 1 min in chlorine at 150 ppm, pH 6.5, within 5 s after inoculation, lesions did not occur. However, if the chlorine treatment was delayed by 120 s, lesions occasionally developed. A 60-min delay usually assured that decay would occur before fruit ripened (within 5 days). Delaying the chlorine wash by 18 h, as would occur if commercial harvests were held overnight prior to packing operations, led to the same incidence of bacterial soft rot as observed among control fruit. Inoculation of fruit with attached stems did not lead to as much decay during storage compared with stemless fruit, suggesting that an intact calyx physically reduced lenticel exposure to external aqueous cell suspensions. Tomato fruit exposed to uncontrolled free moisture during or shortly after harvest have a high potential for postharvest decays and the likely site for infection development is the area surrounding the stem attachment.

Mini-midvastus total knee arthroplasty does not result in superior gait pattern.

PURPOSE: Previous studies dealing with gait after minimally invasive surgery (MIS) total knee arthroplasty (TKA) are rare and insufficient. It was the purpose of the study to determine in a prospective, comparative setting whether MIS influences the outcome of TKA in terms of typical 3D gait parameters. METHODS: Patients scheduled for TKA or MIS TKA were invited to participate. MIS TKA was defined as TKA with shorter skin incision, mini-midvastus arthrotomy, special instruments, and avoidance of tibiofemoral dislocation and patella eversion. All other intra- and perioperative aspects were identical for both groups. A 3D gait analysis was performed with a VICON system 1 month preoperative and 8 weeks post-operative. A multivariate analysis of variance was conducted including the main effects time (pre- and post-surgery) and surgical group and the group-by-time interaction effect. RESULTS: Seventeen MIS TKA patients and 20 TKA patients were eligible for the final analysis. We determined neither inter-group differences nor time × group interactions for any gait variables (temporospatial, ground reaction forces, joint angles and joint moments)­except for the varus-valgus knee kinematics. In pre- to post-operative comparison, the maximum valgus sway increased in the MIS group, whereas it decreased in the conventional group (p = 0.001). CONCLUSION: From our findings, it was concluded that MIS TKA does not result in a superior walking pattern 8 weeks post-operative. Because we previously also observed mini-midvastus MIS TKA to have equal or slightly inferior results with regard to knee scores, knee torque, radiographic outcome and tourniquet/operating time, we discontinued the procedure. LEVEL OF EVIDENCE: Prospective comparative study, Therapy, Level II.

Stage IVB endometrial cancer: clinical course and survival of patients with single and multiple metastases.

OBJECTIVE: Metastatic endometrial cancer (EC) at initial presentation is a rare disease. The present aim was to evaluate prognostic factors and overall survival in patients diagnosed with metastatic EC. STUDY DESIGN: Using data from the Geneva Cancer Registry, the authors included all patients diagnosed with Stage IVB EC from 1980-2007. Estimates of survival were calculated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A total of 38 patients were identified. The most frequent metastases were peritoneal or pleural carcinomatosis (66%, n=25) and hematogenous metastases (53%, n=20). Five-year survival rate was 5.7% (95% confidence interval: 0.0-13.3), and median survival was 7.6 months. Survival of patients with a single metastasis at the time of diagnosis was longer than for patients with multiple metastases (16 versus two months, respectively; p < 0.00 1). CONCLUSION: Metastatic EC is rare disease with very poor prognosis particularly for patients with multiple site metastases.

Community-based case management effectiveness in populations that abuse substances.

BACKGROUND: The number of persons who are substance abusing has been increasing globally. A majority of them remain in their communities, untreated. Empirical studies have shown some positive impacts of case management on substance abuse. However, studies that systematically synthesize the effectiveness of community-based case management with populations that abuse substances are limited. AIM: To review evidence of the impact of case management in improving treatment of substance abuse among adults in community settings. METHODS: The Cochrane processes guided this systematic review. PubMed, CINAHL, PsycINFO, Ovid and the Web of Science were searched to retrieve primary studies published from 2000 to 2013. All randomized controlled trials were considered for review. The methodological quality of the studies was assessed. RESULTS: The initial unfiltered search identified 506 references. A total of seven randomized controlled trials were selected for review. Findings show that, compared with clinical case management and usual care, community-based case management services significantly improved clients' ability to abstain from drug use, reduced social problems, supported unmet service needs and improved satisfaction. Studies also showed reduced use of healthcare services, but results were mixed. CONCLUSIONS: There is an evidence base for practicing case management among adults who are substance abusing. In general, studies concluded that case management is an active and assertive method of care coordination for formal substance abuse treatment. Further research is needed to assess case management's cost-effectiveness and the impact of dosage on client outcomes. IMPLICATIONS FOR NURSING POLICY: Because of the complexity of population health management across settings and over long time frames, evidence-based strategies are required to achieve health improvements. Because it provides continuous and timely care, healthcare leaders and policymakers should consider community-based case management as an important strategy for coordinating the care in populations that are substance abusing.

Winter blood values of selected parameters in a group of non-hibernating captive brown bears (Ursus arctos).

Bears undergo some significant changes reflected in blood values during winter season. The most significant are reduced urea and increased creatinine, by some authors considered to be physiological indicators of hibernation. Studied group of six captive brown bears (Ursus arctos) showed decreased activity in winter but were accepting food and walked outdoors. Blood parameters assessed in February 2011 revealed mean values of leucocytes and neutrophils as significantly lower, and creatinine significantly increased compared to captive and free living bears sampled during other seasons when bears are active.

Resorbable osteosynthetic devices in pediatric traumatology: a prospective series of 24 cases.

UNLABELLED: Bioresorbable devices are commonly used in traumatology. The biomechanical stability of these materials has improved in the past decade, and they have proven to be biologically non-hazardous, while their main advantage is that their use avoids reintervention for removal of the device. A prospective monocentric study was conducted: 24 patients presenting with a fracture that was amenable to osteosynthesis by small-diameter screws were included. These comprised ten tibial spine fractures, four osteochondritis dissecans of the distal femur, eight fractures of the medial epicondyle of the distal humerus, and two distal tibial apophyseal fractures. One or more screws were used that were made of a copolymer of poly-L-lactide-poly-D-lactide acid and trimethylene carbonate with a diameter of 2.8 mm. All patients were immobilized with a cast. Clinical and radiographic monitoring was conducted every month. The entire follow-up protocol had a duration of 24 months. One patient with osteochondritis dissecans presented with joint effusion. Joint stiffness at the time of cast removal resolved completely after 4 months, except for with three children (one epicondyle fracture, two tibial spine fractures). No subjective or objective instability could be detected by clinical examination. Radiographic follow-up revealed no secondary displacement, and all of the fractures had healed. No osteolysis was seen around the screws. No growth disturbances were noticed. Bioresorbable materials thus appear to be a suitable alternative approach for certain pediatric fractures. Their use resulted in outcomes similar to traditional techniques in terms of functional properties and bone healing. Although initial costs are presumably slightly higher, by avoiding a removal operation the total financial burden is most likely reduced. LEVEL OF EVIDENCE: III.

First Experimental Characterization of Microwave Emission from Cosmic Ray Air Showers.

We report the first direct measurement of the overall characteristics of microwave radio emission from extensive air showers. Using a trigger provided by the KASCADE-Grande air shower array, the signals of the microwave antennas of the Cosmic-Ray Observation via Microwave Emission experiment have been read out and searched for signatures of radio emission by high-energy air showers in the GHz frequency range. Microwave signals have been detected for more than 30 showers with energies above 3×10^{16} eV. The observations presented in this Letter are consistent with a mainly forward-directed and polarized emission process in the GHz frequency range. The measurements show that microwave radiation offers a new means of studying air showers at E≥10^{17} eV.

How much does it hurt to be lonely? Mental and physical differences between older men and women in the KORA-Age Study.

OBJECTIVE: Loneliness has a deep impact on quality of life in older people. Findings on sex-specific differences on the experience of loneliness remain sparse. This study compared the intensity of and factors associated with loneliness between men and women. METHODS: Analyses are based on the 2008/2009 data of the KORA-Age Study, comprising 4127 participants in the age range of 64-94 years. An age-stratified random subsample of 1079 subjects participated in a face-to-face interview. Loneliness was measured by using a short German version of the UCLA-Loneliness-Scale (12 items, Likert scaled, ranging from 0 to 36 points). Multiple logistic regression analysis was conducted to analyze the associations of socio-demographic, physical, and psychological factors with loneliness. RESULTS: The mean level of loneliness did not significantly differ between men (17.0 ± 4.5) and women (17.5 ± 5.1). However, among the oldest old (≥85 years), loneliness was higher in women (p value = 0.047). Depression, low satisfaction with life, and low resilience were associated significantly with loneliness, which was more pronounced in men. Living alone was not associated with loneliness, whereas lower social network was associated with a three time higher risk for feeling lonely in both men and women. CONCLUSIONS: The extent of loneliness was equally distributed between men and women, although women were more disadvantaged regarding living arrangements as well as physical and mental health. However, loneliness was stronger associated with adverse mental health conditions in men. These findings should be considered when developing intervention strategies to reduce loneliness.

[Vocational inpatient and post-treatment proposals in cardiac rehabilitation patients (BERUNA): results of a randomized controlled trial]. / Berufsbezogene Rehabilitation und Nachsorge in der kardiologischen Rehabilitation (BERUNA): Ergebnisse einer randomisierten Kontrollgruppenstudie

BACKGROUND: The "Verband Deutscher Rentenversicherungsträger (VDR)" required the intense focus on vocational rehabilitation of medical problems since the early 1990s. By integrating elements of the working environment in the structures and processes of medical rehabilitation, vocational problems can be identified early in order to take appropriate countermeasures. Posttreatment proposals off er a way to provide a reliable long-term success. OBJECTIVE: The intention of this study is the implementation and the investigation of effectiveness of an intensified vocational program that integrates besides the in-patient rehabilitation post-treatment proposals as well. METHODS: Cardiac rehabilitation patient occurring special vocational problems participe. The recruited patients were randomized to either the vocational program or the common cardiac rehabilitation.Primary outcome was the vocational reintegration 12 months after the end of rehabilitation.The database was collected through a query of the pension insurance accounts and questionnaires. RESULTS: 306 people were included to the study at the beginning of rehabilitation. Rates of response:95 % of the survey to the end of rehabilitation,77 % of the survey 6 months and 73 % of the survey 12 months after the end of rehabilitation.The sample was characterized by great interferences of their professional involvement and a high demand for vocational treatments. At the beginning of the rehabilitation 50 % of study participants intended to make an application for reduction-pension. Nevertheless, one year after the end of rehabilitation a surprisingly high rate of vocational reintegration by over 70 % in both groups (IG: 72 %, KG: 75 %, p < 0.929) was indicated.The majority of the unemployed patients at the beginning of rehabilitation remained in the unemployment (IG: 69 %, KG: 65 %, p < 0.757). The participation rate at the post-treatment proposals was 42%. CONCLUSION: The results show that vocational programs in rehabilitation need to be revised, including with regard to the question, whether and in what way post-treatment proposals must be changed.

An integrative review of nurse-led community-based case management effectiveness.

BACKGROUND: Accountable, continuous and patient-centred care in community-based settings is increasingly needed to reduce fragmented care, especially to reduce patient readmission rates. Case management has been proven effective in reducing healthcare costs and in increasing continuity of care, but its effectiveness in community-based settings is not clear. AIM: The aim of this integrative review is to examine the effectiveness of community-based case management programmes in improving patient outcomes. METHODS: An integrative review of articles from PubMed, CINAHL and PsycInfo databases was undertaken to synthesize and analyse research about community-based case management and its outcomes. Studies looking for patient outcomes and empirical studies published since 2000 were included. A total 18 articles were retrieved from 2000 to 2013. RESULTS: Most of the included studies were conducted in the USA, but international studies with quantitative approaches were reviewed (n = 6). The review revealed that community-based case management significantly reduced hospital access outcomes, especially readmissions and increased cost effectiveness, patient clinical outcomes and patient satisfaction. LIMITATIONS: This study did not isolate studies of patients with diseases or demographic characteristics in common, resulting in large variations in disease and demographic factors. CONCLUSION: There is a base of evidence that community-based case management is effective. Because it provides quality, patient-centred care, case management should be used for major care coordination. IMPLICATIONS FOR NURSING AND HEALTH POLICY: The benefits of community-based case management need to be announced to healthcare leaders and policymakers.

Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves.

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.