Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4-CXCR7.

Platelets store and release CXCL12 (SDF-1), which governs differentiation of hematopoietic progenitors into either endothelial or macrophage-foam cells. CXCL12 ligates CXCR4 and CXCR7 and regulates monocyte/macrophage functions. This study deciphers the relative contribution of CXCR4-CXCR7 in mediating the effects of platelet-derived CXCL12 on monocyte function, survival, and differentiation. CXCL12 and macrophage migration inhibitory factor (MIF) that ligate CXCR4-CXCR7 induced a dynamic bidirectional trafficking of the receptors, causing CXCR4 internalization and CXCR7 externalization during chemotaxis, thereby influencing relative receptor availability, unlike MCP-1. In vivo we found enhanced accumulation of platelets and platelet-macrophage co-aggregates in peritoneal fluid following induction of peritonitis in mice. The relative surface expression of CXCL12, CXCR4, and CXCR7 among infiltrated monocytes was also enhanced as compared with peripheral blood. Platelet-derived CXCL12 from collagen-adherent platelets and recombinant CXCL12 induced monocyte chemotaxis specifically through CXCR4 engagement. Adhesion of monocytes to immobilized CXCL12 and CXCL12-enriched activated platelet surface under static and dynamic arterial flow conditions were mediated primarily through CXCR7 and were counter-regulated by neutralizing platelet-derived CXCL12. Monocytes and culture-derived-M1-M2 macrophages phagocytosed platelets, with the phagocytic potential of culture-derived-M1 macrophages higher than M2 involving CXCR4-CXCR7 participation. CXCR7 was the primary receptor in promoting monocyte survival as exerted by platelet-derived CXCL12 against BH3-mimetic induced apoptosis (phosphatidylserine exposure, caspase-3 activation, loss of mitochondrial transmembrane potential). In co-culture experiments with platelets, monocytes predominantly differentiated into CD163(+) macrophages, which was attenuated upon CXCL12 neutralization and CXCR4/CXCR7 blocking antibodies. Moreover, OxLDL uptake by platelets induced platelet apoptosis, like other platelet agonists TRAP and collagen-related peptide (CRP). CXCL12 facilitated phagocytosis of apoptotic platelets by monocytes and M1-M2 macrophages, also promoted their differentiation into foam cells via CXCR4 and CXCR7. Thus, platelet-derived CXCL12 could regulate monocyte-macrophage functions through differential engagement of CXCR4 and CXCR7, indicating an important role in inflammation at site of platelet accumulation.

Tissue plasminogen activator (rt-PA) in acute ischemic stroke: Outcomes associated with ambulation.

PURPOSE: Several clinical trials have highlighted general favorable outcomes of intravenous tissue type plasminogen activator (rt-PA) in acute ischemic stroke using different measures including, National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). Findings from most of these measures indicate that the benefits of rt-PA are time dependent, thus, supporting intensive efforts to fast-track hospital thrombolytic treatment in patients with stroke. Despite the widespread benefits of rt-PA, the effectiveness of this therapy on specific functions such as ambulatory performance of the recovering stroke patient is not fully investigated. We aim to investigate this issue in the current study. METHODS: We analyzed data from a retrospective cohort of acute ischemic stroke patients admitted to Greenville Health System (GHS) between 2010-2013. We identified patients who received rt-PA within a 4.5 hour time frame following the onset of acute ischemic stroke symptoms. Our analysis compared ambulatory variables and hospital-level characteristics in proportions of patients receiving rt-PA with those not receiving rt-PA. This analysis determined whether early treatment with rt-PA is associated with favorable changes in ambulatory status from admission to discharge following acute ischemic stroke. RESULTS: Among 663 patients with ischemic stroke who were eligible to receive rt-PA, 241 patients received rt-PA and 422 patients did not due to several risk factors. We found a statistically significant difference (P < 0.001) for changes in ambulation status from hospital admission to discharge between patients receiving rt-PA and patients who did not receive rt-PA. Among patients who received rt-PA, 27.8% improved in their ambulation status, 41.9% saw no change in their ambulation status, 0.4% worsened in their ambulation status, and 29.9% were unable to be determined. Of the patients who did not receive rt-PA, 20.1% improved in their ambulation status, 61.8% saw no change in their ambulation status, 1.4% worsened in their ambulation status, and 16.6% were unable to be determined. CONCLUSION: Our current study indicates that early treatment with rt-PA may be associated with favorable changes in ambulatory status from admission to discharge following acute ischemic stroke.

Differential MMP-9 activity in CD34⁺progenitor cell-derived foam cells from diabetic and normoglycemic patients.

BACKGROUND: Upon coincubation with platelet aggregates, CD34(+) progenitor cells have the potential to differentiate into foam cells. There is evidence that progenitor cells from diabetic and nondiabetic patients have different properties, which may affect the patients' prognosis. In this study we investigated an in vitro model of foam cell formation based on patient-derived CD34(+) progenitor cells. We analyzed the growth characteristics as well as the M-CSF-release and matrix metalloproteinase (MMP) synthesis from CD34(+) progenitor cell-derived foam cells originating from diabetic and nondiabetic patients. METHODS AND RESULTS: Bone marrow samples were obtained from 38 patients who were elected for thoracic surgery. CD34(+) progenitor cells from diabetic and nondiabetic patients were isolated and incubated with platelets from healthy volunteers. Foam cell formation was confirmed by immunostaining (CD68) and quantified by light microscopy. Whereas the absolute number of foam cells was not affected, the negative slope in the growth curve was seen significantly later in the diabetic group. In supernatants derived from"diabetic" CD34(+) progenitor cells, MMP-9 was significantly enhanced, whereas MMP-2 activity or M-CSF-release was not affected significantly. CONCLUSION: In a coculture model of CD34(+) progenitor cells with platelets, we show for the first time that"diabetic" CD34(+) progenitor cells exhibit functional differences in their differentiation to foam cells concerning growth characteristics and release of MMP-9.

Frailty and osteoporosis in older women--a prospective study.

UNLABELLED: Despite sharing common risk factors and biological pathways, the relationship between frailty and osteoporosis (OP) is not clear. This prospective study has shown that frailty defined by the Vulnerable Elders Survey can predict a decrease in bone mineral density after 1 year. Thus, frail older women should be assessed for osteoporosis. INTRODUCTION: Frailty and OP share common risk factors such as age, sarcopenia, lack of physical activity, low body weight, and smoking. Despite shared risk factors and biological pathways, the relationship between frailty and OP is not clear. The purpose of our prospective study was to examine this relationship in a community sample of older women. METHODS: A sample of 235 community-dwelling women was assessed for demographic, medical, frailty and OP status at baseline, and after at least 1 year. Frailty was assessed using the Cardiovascular Health study (CHS) frailty phenotype and using the Vulnerable Elders Survey (VES-13). OP was measured using dual photon absorptiometry bone mineral density (BMD). Descriptive statistics and regression models were used. RESULTS: At baseline, 235 women with a mean age of 77.6 (SD = 5.4), body mass index (BMI) of 28.3 (SD = 5.2) kg/m(2), and BMD of 0.7 (SD = 0.2) g/cm(2)were assessed. No correlation was found between BMD and the CHS (BMD spine, r = 0.009, p = 0.889; BMD hips, r = 0.050, p = 0.473) or the VES-13 (BMD spine, r = 0.034, p = 0.605; BMD hips, r = -0.042, p = 0.537) frailty scales. One hundred fifty-two (63.9 %) women were assessed after 1 year. In a regression model, women who were frail at baseline (VES-13) were found to have a statistically significantly lower hip and spine BMD at follow-up (controlling for BMI) than women who were non-frail at baseline (p = 0.0393, hip; p = 0.0069, spine). CONCLUSIONS: Frailty status as defined by the VES-13 predicts a decrease in BMD after 1 year.

Are cortical spreading depression and headache in migraine causally linked?

During the past few decades, much controversy has surrounded the pathophysiology of migraine. Cortical spreading depression (CSD) is widely accepted as the neuronal process underlying visual auras. It has been proposed that CSD can also cause the headaches, at least in migraine with aura. We describe three patients, each fulfilling the International Headache Society criteria for migraine with aura, who suffered from headaches 6-10 days per month. Two patients were treated with flunarizine and the third patient with topiramate for the duration of 4 months. All patients reported that aura symptoms resolved completely, whereas the migraine headache attacks persisted or even increased. These observations question the theory that CSD (silent or not) is a prerequisite for migraine headaches.

Outbreak of Salmonella Thompson infection in a Swedish dairy herd.

Salmonella Typhimurium was isolated from a faecal sample from a cow in a Swedish dairy herd after calving. When investigations were undertaken in the herd, Salmonella Thompson was isolated from heifers on a separate pasture, and when these heifers were brought into the herd S Thompson spread rapidly. Control strategies managed to rid the herd of the S Typhimurium infection and the prevalence of S Thompson was at first substantially reduced. There was a rapid increase in its prevalence when the animals were let out to pasture and this development eventually led to the depopulation of the entire herd.

[Ins and outs of neurologic therapy for chronic pain]. / Therapie chronischer Schmerzen in der neurologischen Praxis.

Although chronic pain is common, especially in elderly patients, its treatment often remains inadequate. One of many reasons for this is that insufficient therapy of acute pain carries the risk of making the pain chronic. Sooner or later most patients suffering from chronic pain will consult a neurologist. However, in most neurological departments, pain treatment is neither one of the common medical activities nor a subject in medical education. In Germany, only specialised centres have associated pain outpatient clinics, so it is almost impossible for neurologist trainees to improve their knowledge in pain treatment. This review provides a synopsis of procedures regarding chronic pain treatments, with particular focus on the most frequent pain disorders. The treatment recommendations follow the current guidelines of the German Society of Neurology.

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals.

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.

Comparative analyses of multi-species sequences from targeted genomic regions.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Sows intramammarily inoculated with Escherichia coli influence of time of infection, hormone concentrations and leucocyte numbers on development of disease.

The aim of this study was to identify factors that influence the development of disease in sows inoculated with Escherichia coli in the mammary gland. Ten cross-bred primiparous sows were intramammarily inoculated with living E. coli bacteria at different time points before parturition: seven sows within 48 h before parturition and three sows approximately 96 h before parturition. Before and after inoculation, blood samples and mammary gland biopsy specimens were collected and clinical observations were made. All seven sows inoculated close to parturition developed a rectal temperature of >39.5 degrees C during the first 48 h post-partum and two of them also showed other signs of clinical disease. In the sows inoculated 4 days before parturition, the rectal temperature never exceeded 39.5 degrees C during the first 48 h post-partum and none of them showed any other sign of clinical discase. There was a tendency (P < 0.1) that histological signs of mastitis were more frequent in the sows inoculated close to parturition. There were no overall differences between the two groups of sows in plasma concentrations of cortisol, oestradiol-17beta and 15-ketodihydro-PGF2alpha before inoculation. Before inoculation, the number of neutrophils in the blood was overall higher (P < 0.05) in the group of sows that were inoculated close to parturition. In comparison, the number of lymphocytes before inoculation had a tendency (P < 0.1) to be lower in that group. The data suggest that the time of infection of the mammary gland relative to parturition and the number of circulating neutrophils at the time of infection influence the development of chinical coliform mastitis in the sow.

Specific immune response of mares and their newborn foals to Actinobacillus spp. present in the oral cavity.

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Separate modifiability, mental modules, and the use of pure and composite measures to reveal them.

How can we divide a complex mental process into meaningful parts? In this paper, I explore an approach in which processes are divided into parts that are modular in the sense of being separately modifiable. Evidence for separate modifiability is provided by an instance of selective influence: two factors F and G (usually experimental manipulations) such that part A is influenced by F but invariant with respect to G, while part B is influenced by G but invariant with respect to F. Such evidence also indicates that the modules are functionally distinct. If we have pure measures MA and MB, each of which reflects only one of the parts, we need to show that MA is influenced by F but not G, while MB is influenced by G but not F. If we have only a composite measure MAB of the entire process, we usually also need to confirm a combination rule for how the parts contribute to MAB. I present a taxonomy of separate-modifiability methods, discuss their inferential logic, and describe several examples in each category. The three categories involve measures that are derived pure (based on different transformations of the same data; example: separation of sensory and decision processes by signal detection theory), direct pure (based on different data; example: selective effects of adaptation on spatial-frequency thresholds), and composite (examples: the multiplicative-factor method for the analysis of response rate; the additive-factor method for the analysis of reaction time). Six of the examples concern behavioral measures and functional processes, while four concern brain measures and neural processes. They have been chosen for their interest and importance; their diversity of measures, species, and combination rules; their illustration of different ways of thinking about data; the questions they suggest about possibilities and limitations of the separate-modifiability approach; and the case they make for the fruitfulness of searching for mental modules.

The use of medications for cognitive enhancement.

OBJECTIVE: To provide Canadian physicians and allied health care professionals with the evidence they need to help them make treatment decisions in the management of patients with Alzheimer's disease or other dementias. OPTIONS: The full range and quality of diagnostic and therapeutic modalities available to Canadian physicians for the management of dementia. OUTCOMES: Improvement in the treatment of dementias, leading to reduced suffering, increased functional capacity and decreased economic burden. EVIDENCE AND VALUES: The creation of these evidence-based consensus statements involved literature reviews of the subject by the authors; comparison of alternative clinical pathways and description of the methods whereby published data were analyzed; definition of the level of evidence for data in each case; evaluation and revision in a conference setting (involving primary care physicians, neurologists, psychiatrists, geriatricians, psychologists, consumers and other interested parties); insertion of tables showing key variables and data from various studies and tables of data with recommendations; and reassessment by all authors. BENEFITS, HARMS, AND COSTS: A rational plan for the therapy of dementias is likely to lead to substantial benefits in both human and economic terms. RECOMMENDATIONS: Treatment decisions should be made taking into account the severity or stage of the disease, the availability of caregivers, the presence of disease affecting other bodily systems and the ability of the subject to pay the cost of the medications. Donepezil is considered to have positive effects upon certain tests of neuropsychological function and may produce some improvement in Alzheimer's disease of mild to moderate severity as measured by rating scales. Its ability to improve quality of life remains uncertain. No other drug treatments (apart from symptomatic therapies) are at present approved for the treatment of Alzheimer's disease*. VALIDATION: These recommendations were created by a writing committee, evaluated and revised at a consensus conference and further reviewed and revised by the writing committee prior to publication.

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

Undetected dementia in community-dwelling older people: the Canadian Study of Health and Aging.

OBJECTIVE: To estimate the frequency and correlates of undetected dementia in community-dwelling older people. DESIGN: Secondary analysis of data from the Canadian Study of Health and Aging (CSHA) prevalence survey of dementia. SETTING: All 10 provinces of Canada excluding Indian reserves and military units. PARTICIPANTS: A total of 252 community-dwelling older adults diagnosed with dementia in the CSHA survey. MAIN OUTCOME MEASURE: Undetected dementia, defined as occurring in persons who meet standard diagnostic criteria for dementia but who report never having seen a doctor for memory problems. RESULTS: Of the 252 subjects, 64% had undetected dementia. Subjects with mild functional impairment were significantly more likely to have undetected dementia (odds ratio = 2.4, 95% confidence interval 1.2, 5.0). Older subjects and those with mild cognitive impairment showed a trend toward undetected dementia, although the results did not achieve statistical significance. Educational level, number of comorbid conditions, and degree of social support were not significantly associated with undetected dementia. CONCLUSIONS: A large number of older persons are living in the community with undetected dementia. These older people may be at significant risk for delirium, motor vehicle accidents, medication errors, and financial difficulties. As preventive strategies are developed and new cognitive enhancing therapies emerge, we need to reexamine our current guidelines about screening for cognitive impairment in older adults.

A comparison of colonoscopy and double-contrast barium enema for surveillance after polypectomy. National Polyp Study Work Group.

BACKGROUND: After patients have undergone colonoscopic polypectomy, it is uncertain whether colonoscopic examination or a barium enema is the better method of surveillance. METHODS: As part of the National Polyp Study, we offered colonoscopic examination and double-contrast barium enema for surveillance to patients with newly diagnosed adenomatous polyps. Although barium enema was performed first, the endoscopist did not know the results. RESULTS: A total of 973 patients underwent one or more colonoscopic examinations for surveillance. In the case of 580 of these patients, we performed 862 paired colonoscopic examinations and barium-enema examinations that met the requirements of the protocol. The findings on barium enema were positive in 222 (26 percent) of the paired examinations, including 139 of the 392 colonoscopic examinations in which one or more polyps were detected (rate of detection, 35 percent; 95 percent confidence interval, 31 to 40 percent). The proportion of examinations in which adenomatous polyps were detected by barium enema colonoscopy was significantly related to the size of the adenomas (P=0.009); the rate was 32 percent for colonoscopic examinations in which the largest adenomas detected were 0.5 cm or less, 53 percent for those in which the largest adenomas detected were 0.6 to 1.0 cm, and 48 percent for those in which the largest adenomas detected exceeded 1.0 cm. Among the 139 paired examinations with positive results on barium enema and negative results on colonoscopic examination in the same location, 19 additional polyps, 12 of which were adenomas, were detected on colonoscopic reexamination. CONCLUSIONS: In patients who have undergone colonoscopic polypectomy, colonoscopic examination is a more effective method of surveillance than double-contrast barium enema.

The genomic region encompassing the nephropathic cystinosis gene (CTNS): complete sequencing of a 200-kb segment and discovery of a novel gene within the common cystinosis-causing deletion.

Nephropathic cystinosis is an autosomal recessive disorder caused by the defective transport of cystine out of lysosomes. Recently, the causative gene (CTNS) was identified and presumed to encode an integral membrane protein called cystinosin. Many of the disease-associated mutations in CTNS are deletions, including one >55 kb in size that represents the most common cystinosis allele encountered to date. In an effort to determine the precise genomic organization of CTNS and to gain sequence-based insight about the DNA within and flanking cystinosis-associated deletions, we mapped and sequenced the region of human chromosome 17p13 encompassing CTNS. Specifically, a bacterial artificial chromosome (BAC)-based physical map spanning CTNS was constructed by sequence-tagged site (STS)-content mapping. The resulting BAC contig provided the relative order of 43 STSs. Two overlapping BACs, which together contain all of the CTNS exons as well as extensive amounts of flanking DNA, were selected and subjected to shotgun sequencing. A total of 200,237 bp of contiguous, high-accuracy sequence was generated. Analysis of the resulting data revealed a number of interesting features about this genomic region, including the long-range organization of CTNS, insight about the breakpoints and intervening DNA associated with the common cystinosis-causing deletion, and structural information about five genes neighboring CTNS (human ortholog of rat vanilloid receptor subtype 1 gene, CARKL, TIP-1, P2X5, and HUMINAE). In particular, sequence analysis detected the presence of a novel gene (CARKL) residing within the most common cystinosis-causing deletion. This gene encodes a previously unknown protein that is predicted to function as a carbohydrate kinase. Interestingly, both CTNS and CARKL are absent in nearly half of all cystinosis patients (i.e., those homozygous for the common deletion). [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF168787 and AF163573.]

Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.

Effects of Actinobacillus equuli culture supernatants on equine neutrophil functions and survival.

After exposure of equine granulocytes from both foals and adult horses to culture supernatants from clinical isolates of Actinobacillus equuli, phagocytic capacity and respiratory burst was examined by flow-cytometry and a chemiluminescence assay, respectively. One haemolytic isolate of an equine Actinobacillus was also included in the study. An average decrease of 22% in total number of granulocytes, in the flow cytometric assay (P < 0.01), and an average decrease of 26% in light emission, in the chemiluminescence assay (P < 0.001), was seen after exposure to bacterial culture supernatants of A. equuli, indicating that the supernatants contained leukotoxic bacterial products. Supernatants from the haemolytic isolate appeared to contain a higher amount or more potent leukotoxic metabolites when haemolysis was expressed, causing a decrease in total number of granulocytes of 44% (P < 0.01) and a decrease in light emission of 52% (P < 0.01). Evaluation of the stability of the methods used revealed that within-method variation was far less than the observed results. The leukotoxic effects of A. equuli culture supernatants were mainly reflected in the decreased survival of neutrophils and not in neutrophil functions.