Biting the hand that feeds: Anthropogenic drivers interactively make mosquitoes thrive.

Anthropogenic stressors on the environment are increasing at unprecedented rates and include urbanization, nutrient pollution, water management, altered land use and climate change. Their effects on disease vectors are poorly understood. A series of full factorial experiments investigated how key human induced abiotic pressures, and interactions between these, affect population parameters of the cosmopolitan disease vector, Culex pipiens s.l. Selected pressures include eutrophication, salinity, mean temperature, and temperature fluctuation. Data were collected for each individual pressure and for potential interactions between eutrophication, salinization and temperature. All experiments assessed survival, time to pupation, time to emergence, sex-ratio and ovipositioning behavior. The results show that stressors affect vector survival, may speed up development and alter female to male ratio, although large differences between stressors exist to quite different extents. While positive effects of increasing levels of eutrophication on survival were consistent, negative effects of salinity on survival were only apparent at higher temperatures, thus indicating a strong interaction effect between salinization and temperature. Temperature had no independent effect on larval survival. Overall, increasing eutrophication and temperatures, and the fluctuations thereof, lowered development rate, time to pupation and time to emergence while increasing levels of salinity increased development time. Higher levels of eutrophication positively impacted egg-laying behavior; the reverse was found for salinity while no effects of temperature on egg-laying behavior were observed. Results suggest large and positive impacts of anthropogenically induced habitat alterations on mosquito population dynamics. Many of these effects are exacerbated by increasing temperatures and fluctuations therein. In a world where eutrophication and salinization are increasingly abundant, mosquitoes are likely important benefactors. Ultimately, this study illustrates the importance of including multiple and combined stressors in predictive models as well as in prevention and mitigation strategies, particularly because they resonate with possible, but yet underdeveloped action plans.

Concordance in KRAS and BRAF mutations in endoscopic biopsy samples and resection specimens of colorectal adenocarcinoma.

BACKGROUND: KRAS testing is mandatory if anti-EGFR therapy is considered in patients with metastatic colorectal cancer (CRC). In addition, BRAF mutations seem to be an important negative prognostic factor. The aim of this study is to establish the concordance of KRAS and BRAF mutational status in paired biopsy and resection specimens of primary CRC using several analytic methods. METHODS: DNA was extracted from paraffin blocks of 126 CRC patients. KRAS codon 12/13 and BRAF V600E mutational status was assessed using high resolution melting (HRM), direct sequencing (DS) of the HRM polymerase chain reaction (PCR) product. In addition, the Therascreen Amplification Refractory Mutation System (ARMS)-Scorpion KRAS assay and BRAF pyrosequencing were employed; both assays claim to require less tumour cells in comparison with DS. RESULTS: KRAS and BRAF were found to be mutually exclusive. Mutation frequencies were 33.9% for KRAS, and for BRAF 19.0%, respectively. Concordance of KRAS mutational status between biopsy and resection specimens was 97.4% (ARMS), 98.4% (DS) and 99.2% (HRM), respectively. For BRAF concordance was 98.4% (Pyro, DS) and 99.2% (HRM). CONCLUSIONS: KRAS and BRAF mutational status of endoscopic biopsies and resection specimens of CRC showed a >95% concordance. Endoscopic biopsies can be confidently used for molecular analysis.

Danon disease: a focus on processing of the novel LAMP2 mutation and comments on the beneficial use of peripheral white blood cells in the diagnosis of LAMP2 deficiency.

Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients. Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4+ and CD8+ T lymphocytes, CD20+ B lymphocytes, CD14+ monocytes and CD56+ natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8+ T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells. The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT.

Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic SCT (HSCT) frequently results in CMV reactivation, which may in turn lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4+ and CD8+ T-cells in 191 samples from 118 individuals. We compared healthy donors with both patients with high and undetectable viral loads, and those who controlled and did not control their CMV reactivations. Polychromatic flow-cytometric measurements of CD154 (CD40L), intracellular cytokines (IFNγ, IL2) and a degranulation marker (CD107a) allowed us to assess the functional status of various T-cells simultaneously. We found that dual IFNγ/IL2-producing CD8+ T-cells were present in patients controlling their CMV reactivations but absent from non-controllers. CD8+ T-cells that produce only IFNγ were the most abundant subtype, but they most likely represent non-protective memory cells. Distinct functional signatures were examined by hierarchical clustering, and this revealed that, unlike polyfunctional CD8+ T-cells, CD8+ T-cells that produce IFNγ alone were not functioning in concert with other subsets. In conclusion, our study revealed functional signatures that may be useful for immune monitoring, and it could change the interpretation of previous studies that assessed only IFNγ.

Prognosis of pediatric patients transplanted for Ph+ chronic myeloid leukemia in the period from 1989 to 2006 in the Czech Republic.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by clonal proliferation of primitive hematopoietic stem cell. The median age at diagnosis is 55 to 60 years with less than 10% of patients younger 20 years. Incidence of CML in children in the Czech Republic is 0.106 cases/100 thousands per year. Here we report outcome of 38 pediatric patients (median age 12.5 years; range 1.8 - 17.3) with Ph-positive CML diagnosed between years 1989 to 2006. Primarily chronic phase of the disease was diagnosed in 32 (84%) patients. 32 (84.2%) patients underwent hematopoietic stem cell transplantation (HSCT) with the median age at transplantation of 14.9 years (range 6.9 - 20.5 years). Out of transplanted patients 16 (50%) obtained graft from unrelated donor, 13 (41%) from matched sibling donor, 2 from haploidentical family donor and autologous transplantation has been performed in one case. 6 patients were not transplanted, 4 of them died (median 1.2 years from diagnosis), 2 are alive 0.6 and 17.8 years from the diagnosis. Overall survival (OS) in 25 patients after HSCT at our department during the whole period is 66.7% with 15/16 being in stable continuous molecular-genetic remission (94%). During the period of time results of transplantations have been significantly improved (p=0.0071). OS after HSCT until year 1997 is 25% while from year 1998 until now is 87.5%. All centers OS of patients after HSCT is 71%. Results of HSCT in children with CML obtained from the year 1998 at our center are fully comparable with results achieved in large and experienced centers. HSCT remains the only proven and effective method for the treatment of CML. Clinical studies assessing the role of tyrosine kinase inhibitors in children instead of early HSCT should be planned carefully in order to avoid sub-optimal outcomes.

Low mortality of children undergoing hematopoietic stem cell transplantation from 7 to 8/10 human leukocyte antigen allele-matched unrelated donors with the use of antithymocyte globulin.

Human leukocyte antigen (HLA)-matched sibling donor hematopoietic stem cell transplantation (HSCT) is available for only approximately 30% patients needing HSCT. Use of alternative donors is associated with a high incidence and severity of graft-versus-host disease (GVHD). Here we report our experience with GVHD prophylaxis using pre-transplant rabbit antithymocyte globulin (rATG), in addition to post transplant cyclosporin A and methotrexate. Seventy-five children received unmanipulated grafts from 7 to 10/10 HLA allele-matched unrelated donors. Median follow-up was 25 months (range, 6-65 months). Only 2/75 patients (2.5%) developed acute GVHD grades III-IV, and 17/75 (25%) developed extensive chronic GVHD. Overall survival was 79%. It was similar in patients receiving grafts from 7 or 8/10 to 9 or 10/10 allele-matched donors, and similar in patients receiving peripheral blood stem cells and marrow. Six (11%) patients died owing to relapse, and 10 (13%) due to transplant-related complications. The addition of rATG appears to result in a low incidence of severe GVHD and overall mortality.

Age rationing for renal transplantation? The role of age in decisions regarding scarce life extending medical resources.

The use of age as a selection criterion for scarce life extending medical resources is justified by some theorists and rejected by others. Qualitative research was conducted into age rationing in daily medical practice. Observations were made at two renal transplantation centres and people professionally involved in decision making about transplantation were interviewed. Age appeared to be an important factor in indication decisions concerning individual patients, because it is associated in several ways with both the risks and benefits of transplantation that are weighed against each other. This happens apart from scarcity of donor organs. However, age also appeared to be used as a selection criterion, though apparently to a slight degree. This happens in a covert, implicit way. This is possible because all the aspects of age that are important in indication decisions regarding individual patients may also be used as comparative selection criteria.

The meaning of urgency in the allocation of scarce health care resources; a comparison between renal transplantation and psychogeriatric nursing home care.

In the juridical and ethical literature on patient selection criteria it is an unargued premise that those who are most urgently in need of treatment or care will be given priority. The aim of this study is to gain insight into the medical practice of waiting list problems and patient selection at the microlevel, especially with respect to urgency. Thus, the study intends to contribute to the medical ethical discussion on patient selection for scarce resources. The results of qualitative research into the meaning and occurrence of urgency in two health care services, renal transplantation and psychogeriatric nursing home care, are discussed. In the first sector, patients are seldom considered urgent. Criteria for urgency are technical dialysis problems or severe psychological burden due to protracted dialysis treatment. In contrast, psychogeriatric patients are often considered urgent, with the principal criterion being too heavy a care load for informal carers. Both health care services show variation in assigning urgency codes. It appears that the exact meaning of urgency is not self-evident and that admission of urgent patients to nursing homes can be negotiated by professionals or informal carers. This points to the necessity of a discussion within these services as to the actual content matter of urgency. Further, professionals involved in renal transplantation raise several moral and practical arguments against giving patients priority, even if they need treatment urgently. It shows that distributive justice cannot always be applied. Occasionally non-urgent patients are rated urgent as they have been waiting very long due to specific allocation procedures. In these cases urgency is granted in an unexpected way that is ultimately in accordance with the notion of procedural justice.

[Selection of patients for kidney transplantation; how to deal with scarcities in clinical practice]. / Selectie van patiënten voor een niertransplantatie; hoe met schaarste in de medische praktijk wordt omgegaan.

OBJECTIVE: To investigate what criteria are applied in the actual practice of patient selection for renal transplantation and how practice relates to guidelines developed in medical ethics and health law. SETTING: Two centres for renal transplantation and nine dialysis centres. DESIGN: Descriptive. METHODS: Data were collected by observation and open interviews with 33 physicians and other health professionals involved, and analysed using a computer programme for qualitative data. RESULTS: Formally, scarcity of donor organs did not affect the referral and indication of patients for renal transplantation. However, according to some respondents, fewer people were entered on the waiting list because of scarcity. This concerned mainly patients less likely to benefit from a transplantation in terms of life expectancy or enhancement of quality of life. CONCLUSION: There was some 'covert selection', in that scarcity implicitly or unintentionally was a factor in the decision whether or not to place patients on the waiting list for renal transplantation. The absence of consensus on acceptable selection criteria and the emphasis on medical criteria in the social debate on selection criteria may have contributed to this covert selection.

The emergency department management of near-hanging victims.

A 7-year retrospective review was performed to assess the complications of near-hangings injuries. Thirty-nine cases of near hanging were seen during this period. There were no hanging drops greater than 5 feet and no cervical spine fractures. One patient required intubation for soft tissue swelling. The adult respiratory distress syndrome (ARDS) occurred in three patients. All victims with field Glasgow Coma Scale levels >3, and three of eight with GCS = 3 survived to discharge with a normal mental status. We conclude that aggressive resuscitation and treatment of postanoxic brain injury is indicated even in patients without evident neurologic function in the field, as full recovery may still occur. Cervical spine fractures have not been reported in near-hanging victims and should only be considered if there is a possibility of a several foot drop or if a focal neurologic deficit is present. Injury to the anterior soft tissues of the neck may cause respiratory obstruction. Close attention to the development of pulmonary complications is required.

Attitudes of health-care workers towards AIDS at three Dutch hospitals.

A questionnaire survey was held among 938 doctors and 2304 nurses to assess their attitudes toward AIDS and the influence of their concern about the occupational risks involved. The response was 65 and 72%, respectively. The results suggest that in treating patients with actual or possible HIV infection, in non-invasive procedures many doctors and nurses often take too many precautions, whereas in invasive procedures doctors often take too few. A minority of the respondents were in favour of testing all patients. The majority felt that patients in the high-risk groups should be tested. The percentage in favour of anonymous testing was considerably higher among the doctors than among the nurses. Most of the doctors and nurses were concerned about contagion by patients. This concern had a negative influence on their attitudes toward AIDS. Factual information alone does not suffice to dispel excessive concern. In training and educating medical personnel, attention should be devoted to cognitive as well as emotional aspects.

[Parental visiting and involvement in the neonatal intensive care unit]. / Bezoek en participatie van de ouders op de couveuse-afdelingen.

Out of 145 neonatal departments of Dutch hospitals 120 participated in an inquiry concerning parental visiting and involvement. The results point to the conclusion that involvement of the parents is a fact that concerns practically all departments. Parents are allowed much more than some ten years ago. However, talks with parents reveal that many of them are not satisfied by far concerning these matters. Therefore the value of this kind of study is in revealing social desirability rather than actual reality.

[Referral: the meeting point between primary and secondary care]. / Verwijzing: ontmoetingspunt tussen 1e en 2e lijn.

The growing number of referrals to specialists is a matter of concern to the Government and the 'health insurance collective' (ziekenfonds). In spite of all kinds of measures this increase in referrals continues, also in pediatrics. An effective policy to reverse this trend depends on an understanding of its causes. This paper goes into the mechanisms of supply and demand in health care and the roles of both G.P. and patient in the decision to refer. Parents worry about their children's symptoms. These worries are not always understood by their general practitioner and may lead to referrals in which not medical questions but the worries of the parents take a central place. In treatment at the Out-Patient Clinic consideration should be given not only to the questions, worries and doubts of the G.P., but also to those of the patient and above all the patient's parents.