Complications of whole abdominal and pelvic radiotherapy following chemotherapy for advanced ovarian cancer.

We examined the records of 105 patients with advanced ovarian cancer who had been treated with cisplatin combination chemotherapy followed by abdominopelvic radiotherapy. The purpose was to define the morbidity of this approach, and identify those factors predictive of toxicity. Acute toxicity resulting in delay or failure to complete treatment was most commonly due to myelosuppression. Nine of 105 patients (8.6%) required surgery for bowel obstruction that was not due to recurrent disease, 3 had an episode of bowel obstruction that settled conservatively, and a further 5 underwent surgery for obstruction due to recurrent tumor. The presence of both a dose of abdominopelvic radiotherapy over 2250 cGy, as well as a second-look laparotomy prior to radiotherapy, was associated with an increased risk of serious bowel complications. The increased frequency of late bowel morbidity seen in the combined modality group is likely explained by the presence of these two factors, rather than the exposure to chemotherapeutic agents per se. These observations are supported by the published literature.     

Corticofugal modulation of the information processing in the auditory thalamus of the cat

Summary Single unit activity of 355 cells was recorded in the auditory thalamus of anesthetized cats before, during, and after the inactivation by cooling of the ipsilateral primary auditory cortex (AI). Most of the units (n = 288) showed similar functional characteristics of firing before and after the cryogenic blockade of AI. The spontaneous firing rate remained unchanged by cooling in 20% of the units and decreased in the majority of them (60%). In some regions, i.e. dorsal division of the medial geniculate body (MGB), lateral part of the posterior group of the thalamus, and auditory sector of the reticular nucleus of the thalamus, the maximum firing rate evoked by white noise bursts was generally affected by cooling in the same direction and to the same extent as the spontaneous activity. Units in the ventral division of MGB showed a characteristic increase of signal-to-noise ratio during cortical cooling. The corticofugal modulation led to the appearance or disappearance of the best frequency of tuning in 51 units and changed it by more than 0.5 octave in 34 units. The bandwidths of different response patterns to pure tones stimulation were used to define a set of functional properties. During cryogenic blockade of AI, two cortically modulated sub-populations of units were usually distinguished that exhibited changes for a given functional property. The complexity and diversity of the effects of cortical inactivation suggest that the corticothalamic projection may be the support for selective operations such as an adaptive filtering of the incoming acoustic signal at the thalamic level adjusted as a function of cortical activity.     

Analysis of complications in patients treated with abdomino-pelvic radiation therapy for ovarian carcinoma.

Between 1971 and 1985, 598 patients with ovarian carcinoma were treated with abdomino-pelvic radiation therapy. Acute complications included nausea and vomiting in 364 patients (61%) which were severe in 36, and diarrhea in 407 patients (68%), severe in 35. Leukopenia (less than 2.0 x 10(9) cells/liter) and thrombocytopenia (less than 100 x 10(9) cells/liter) occurred in 64 patients (11%) each. Treatment interruptions occurred in 136 patients (23%), and 62 patients (10%) did not complete treatment. In both situations the most common cause was myelosuppression. Late complications included chronic diarrhea in 85 patients (14%), transient hepatic enzyme elevation in 224 (44%), and symptomatic basal pneumonitis in 23 (4%). Serious late bowel complications were infrequent: 25 patients (4.2%) developed bowel obstruction and 16 required operation. Multivariate analysis was unable to determine any significant prognostic factors for bowel obstruction; however, the moving-strip technique of radiation therapy was associated with a significantly greater risk of developing chronic diarrhea, pneumonitis, and hepatic enzyme elevation than was the open beam technique. We conclude that abdomino-pelvic radiation therapy as used in these patients is associated with modest acute complications and a low risk of serious late toxicity.     

Regulation of cytokine synthesis in cardiac surgery: Role of extracorporeal circuit and humoral mediators <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis>

Objective and design: Cardiopulmonary bypass (CPB) impairs monocyte and neutrophil proliferation, cytokine synthesis, and antigen presentation. This study compares in vivo data with results from an extracorporeal circulation (ECC) model, distinguishing direct effects on cytokine synthesis from regulatory mechanisms. Patients and methods: Whole blood from 18 patients prior to, during and after CPB was stimulated with lipopolysaccharide (LPS). Tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 levels were measured. Additionally, blood from 4 volunteers was circulated in an ECC model. Cytokine levels were measured before and during mock ECC. Results: LPS-induced cytokine synthesis was reduced after CPB (TNF-α: 11 %; IL-6: 29 %; IL-8: 48 % of preoperative values, all p < 0.001). In mock ECC, cytokine production (except IL-8) was suppressed: TNF-α production was lowest 60 min after starting ECC, IL-6 synthesis was lowest at 90 min (33 % and 15 % vs. pre-ECC levels; both p < 0.001). Patient sera contained cytokine-inhibitory activity after CPB, an activity not found in mock ECC. Conclusions: (1) In patients, CPB induces early transient LPS hyporesponsiveness; (2) blood contact with foreign surfaces induces LPS hyporesponsiveness; (3) serum cytokineinhibitory activities are released after CPB, but not in mock ECC. Impaired leukocyte function may explain increased susceptibility to infections after CPB. Received 16 September 2006; accepted without revision by K. Visvanathan 18 October 2006     

Oncogene-associated transformation of rodent fibroblasts is accompanied by large morphologic and metabolic alterations

Cellular growth, proliferative activity, cell volume and metabolism of four differently transformed cell lines were investigated. Studies were carried out with spontaneously immortalized and poorly tumorigenic Rat1 cells, c-mycl-transfected and non-tumorigenic M1 fibroblasts, as well as their T24Ha-ras-(co)-transfected counterparts Rat1-T1 and MR1. Ras-transfection of both Rat1 and M1 cells, which is associated with aggressive tumor growth in vivo, caused significant morphological alterations, namely a 30-50% decrease in cell volume. A negative linear correlation between cell number and cell volume at confluence was observed. Furthermore, the expected stimulation of cellular growth rate after T24Ha-rastransfection was documented. Growth inhibition in Rat1 and M1 cultures was reflected by a dramatic decrease in the [H-3]thymidine labeling index (TLI) to below 3% while entering the stationary growth phase. In contrast, Rat1-T1 and MR1 cells had a TLI of greater than or equal to 18% even at confluence. Glucose uptake and lactate production were not different on a per cell basis: between parental cell line and the T24Ha-ras-transfected transformants. However, when these parameters were normalized for differences in cell volume, ras-transfection-resulted in increased glucose consumption and lactate release. The behavior of cellular and cell volume-related oxygen uptake throughout the growth period examined was remarkably different between the parental lines and the ras-transformed descendants. Oxygen consumption rates (QO(2)) of Rat1-T1 and MR1 cells were significantly less than those of Rat1 and M1 fibroblasts and showed different changes as a function of time in monolayer culture. Whereas the cellular QO(2) of the highly tumorigenic cell clones either decreased or leveled off throughout the entire period of plateau phase, a decline in cellular oxygen uptake was observed in the partly transformed cells until days 4-5 only. The decline was then followed by an increase with values almost returning to those recorded during the early exponential phase. The data presented demonstrate for the first time an impact of well-defined oncogenic alterations on the metabolic characteristics of cells which is a further step in the understanding of the pathophysiology of ras-associated tumor cells.     

An optimized fast protocol for magnetic resonance imaging of the temporomandibular joint

OBJECTIVES: To provide a fast protocol for MR imaging of the TMJ with high contrast resolution of both soft tissue and joint fluid. METHODS: A fast turbo-spin echo (TSE) pulse sequence was developed. The new technique was compared with T1W conventional spin echo (CSE) and T2W TSE sequences in imaging 57 TMJs in 50 patients. Disc location and contour and bone delineation were assessed on three images of each TMJ by one observer. Presence and location of intra-articular fluid were evaluated on both standard T2W and new TSE images. Image quality was scored on a 3 point-scale by one observer. RESULTS: The net image acquisition time for one plane was 2 min with the new TSE sequence compared with approximately 5-10 min for T1W and 4 min for T2W. The new sequence provided equivalent diagnostic information to a combination of T1W CSE for disc position and contour and bone contour and T2W TSE sequence for joint fluid. CONCLUSIONS: The examination time for MRI of the TMJ can be considerably reduced with the new fast TSE pulse sequence without compromising image quality.     

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Chickens and pigs are the most important livestock species worldwide. They are not only important sources of food, but also valuable models for evolutionary biology and biomedical science. Pigs share a high anatomical and physiological similarity with humans and are an important species for translational biomedical research, for example, in the areas of cancer, diabetes, neurodegenerative, and cardiovascular diseases (1–3). They also resemble the human pathophenotype more closely than rodents. For example, pig models for familial adenomatous polyposis (FAP) develop polyps in the large intestine as observed in human patients (4), whereas mouse FAP models develop them in the small intestine (5). In contrast to mammals, chickens are phylogenetically distant vertebrates from humans, but they were instrumental in the field of developmental biology due to the easy access to the embryonated egg. They are used for studying neurological and cardiovascular functions (6–8) and provided key findings in B cell development and graft versus host responses (9–11). Genetically modified livestock species also hold great promise for agriculture by offering new approaches for disease control, such as genome-edited pigs resistant to Porcine Reproductive and Respiratory Syndrome or Avian Leucosis Virus (ALV)-resistant chickens (12–15).Due to the lack of fully functional embryonic stem cells, genetic engineering in pigs and chickens has been a laborious, inefficient, and time-consuming procedure (16). The generation of pigs with precise germline modifications required gene targeting in somatic cells followed by somatic cell nuclear transfer. This also is not practical in chickens, where precise alteration of the genome only became possible with recent improvements in the cultivation and manipulation of germline-competent primordial germ cells (PGCs) (17–19). These modified PGCs can be injected into the blood vessel system of stage 13 to 15 (Hamburger−Hamilton [HH]) embryos to produce germline chimeras and, by further breeding, genetically modified chickens.With the advent of synthetic endonucleases such as CRISPR-Cas9 efficiency of targeted germline modification has improved in both species (20–23). It still requires the generation and breeding of new founder lines, which is time consuming in large animals. To circumvent the need for generating germline-modified animals, attempts have been made to carry out genome editing directly in specific organs or tissues (24–27). But this has been hampered by the need to deliver both Cas9 and the required guide RNA (gRNA) and by the limited cargo capacity of viral vectors. To bypass this drawback, Cas9 transgenic mice have been generated, requiring delivery of only the respective gRNAs (28).Here, we describe the generation of both Cas9 transgenic pigs and chickens that ubiquitously express Cas9 endonuclease and provide proof of its function in vitro and in vivo. These animals provide an innovative and efficient model for in vivo genome editing to assess gene function in health and disease.     

Childhood insulinoma masquerading as seizure disorder

A 9 year old girl presented with seizures, weight gain and early morning behavioural changes. She had been commenced on anticonvulsants and was subsequently diagnosed with hyperinsulinaemic hypoglycaemia. This case demonstrates the importance of blood glucose monitoring in children presenting with new‐onset seizures and/or with early morning or fasting behavioural changes, the challenges in localizing the lesion, as well as the difficulties in achieving normoglycaemia prior to, and immediately following, surgery.     

Sequence and genome context analysis of a new molecular class D beta-lactamase gene from Legionella pneumophila

Legionella pneumophila Philadelphia-1 (ATCC 33152) produces a serine active site beta-lactamase. The chromosomal gene that encodes this enzyme, loxA, has been cloned by PCR using information from the L. pneumophila Philadelphia-1 genome sequencing project. LoxA is a class 2d penicillinase, and its sequence puts it into the molecular class D beta-lactamase family, although phylogenetic analysis shows that LoxA forms a distinct branch in the OXA family along with the LoxA homologue, OXA-29, from Legionella gormanii ATCC 33297(T). Upstream of loxA on the L. pneumophila Philadelphia-1 chromosome is a two-gene locus similar to that found linked to the beta-lactamase genes of Gram-positive bacteria. The unit consists of loxI, encoding a homologue of the Gram-positive beta-lactamase expression regulator, and pbpX, encoding a putative penicillin-binding transpeptidase. Despite the presence of beta-lactamase regulator homologues, we could find no evidence of LoxA induction upon challenge of L. pneumophila Philadelphia-1 with beta-lactams.     

Glycation of extracellular matrix proteins impairs migration of immune cells

The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes‐related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T‐cells. To achieve our purpose, we used in vitro AGE‐modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T‐cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin‐Alexa‐fluor 488‐labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE‐bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE‐modified matrix, but not with soluble AGEs like BSA‐AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs‐modified matrix protein inhibits cell migration and adhesion of Jurkat T‐cells.