Benefits of spinal anesthesia for urologic surgery in the youngest of patients.

INTRODUCTION: Increasing concerns regarding potential negative effects of early use of inhalational and intravenous anesthetics on neurocognitive development have led to a growing interest in alternative forms of anesthesia in infants. The study institution's outcomes with spinal anesthesia (SA) for urologic surgery in infants aged less than 90 days are reported and their outcomes with a matched cohort of patients who underwent general anesthesia (GA) are compared. METHODS: This is a retrospective single-center analysis. Patients aged less than 90 days who underwent SA for four urologic surgeries (inguinal hernia repair, scrotal exploration, posterior urethral valve ablation, and ureterocele puncture) were identified from the study institution's SA database. An age- and procedure-matched control cohort was identified from a list of patients who underwent the aforementioned four procedures under GA since 2013. Outcomes of interest included success rate of SA, complications from spinal placement, narcotic use, need for supplemental medications and oxygen, and length of hospital stay. RESULTS: Forty patients were identified; 20 in the SA and 20 in the GA group. Mean patient age was 54 (standard deviation, 35) days. There were no significant differences between the groups in age, gender, weight, history of prematurity, or presence of comorbidities. Eighty percent of SA patients had successful SA; reasons for conversion to GA included failure of spinal needle placement (75%) and agitation during operative procedure (25%). Ninety-six percent of patients who received GA (primarily or converted) had an endotracheal tube (ETT) placed. No patient in the SA group had a complication from spinal needle placement. Patients in the SA group were less likely to receive narcotics during the operative procedure (P = 0.001) and also had a lower mean morphine equivalent dose/kilogram (P = 0.002). Patients in the SA group were also less likely to receive any supplemental medications during the operative procedure (P = 0.001), particularly intravenous corticosteroids (P < 0.001). There were no significant differences in the length of hospital stay. CONCLUSIONS: The use of SA has clear advantages for this medically vulnerable population. For the majority of patients, it obviates the need for ETT placement and airway management and avoids the potential negative effects of GA on neurocognitive development. It also decreases the use of narcotics and other supplemental medications. In scenarios in which the benefit of surgery must be weighed against the risk of GA, such as neonatal torsion, SA may allow a paradigm shift in the timing of surgery.

Contaminated area instability along Ångermanälven River, northern Sweden.

Industrially utilized river basins are frequently exposed to contaminants originating from polluting activities. However, the physical instability and probability of mass movement mobilization of contaminated soil into rivers have only received little attention. In this study, we present a GIS-based method to produce a regional overview of where and how contaminated areas are potentially exposed to slope instability. A landslide susceptibility-index was used to study the degree and distribution of overlap between contaminated sites and unstable ground. A contaminated area instability hazard classification was produced integrating slope instability and contamination risk classification. Our results indicate that mass movement can be tied mainly to a slope gradient ≥16°, a proximity to the river that is <500 m, a distance of <500 m from roads, concave surface curvature, and sand- and silt soils. Forty-six (22%) of all considered contaminated sites are located within areas with a non-negligible slope instability, of which a majority, 30 sites (14%) are situated on ground with a low or moderate instability. Three sites with a class 2 contamination risk (the 2nd highest class) are located on ground with a very high slope instability.

Molecular characterisation of foot-and-mouth disease viruses from Pakistan, 2005-2008.

Foot-and-mouth disease (FMD), an economically important disease of cloven-hoofed animals, is endemic in Pakistan where three virus serotypes are present (O, A and Asia 1). Fifty-eight clinical samples collected between 2005 and 2008 from animals with suspected FMD in various locations in Pakistan were subjected to virus isolation on primary cell culture, antigen ELISA and real-time RT-PCR (rRT-PCR). Viruses were isolated from 32 of these samples and identified as FMDV type O (n = 31) or type A (n = 1). Foot-and-mouth disease virus (FMDV) genome was detected in a further 11 samples by real-time RT-PCR. Phylogenetic analyses of the VP1 nucleotide sequences showed that all of the type O viruses belonged to the MIDDLE EAST-SOUTH ASIA topotype with the majority belonging to the PanAsia-2 lineage; a single example of the older PanAsia lineage was identified. The single FMDV type A virus belonged to the ASIA topotype, but did not cluster with known strains that are currently circulating (such as Iran-05) and was not closely related to other type A viruses from the region. These findings demonstrate the widespread distribution of O-PanAsia-2 in Pakistan and the presence of undisclosed novel type A lineages in the region.

Molecular characterization of foot-and-mouth disease viruses collected from Sudan.

The aim of this study was to characterize foot-and-mouth disease (FMD) viruses collected between 2004 and 2008 from Sudan, a country where FMD is endemic. Using virus isolation and antigen ELISA, three FMD virus serotypes (O, A and SAT2) were detected in 24 samples that were submitted to the FAO World Reference Laboratory for FMD. Pan-serotypic real-time RT-PCR assays targeting the 5' untranslated region (5'UTR) and 3D genes of FMD virus were also used to contribute to the laboratory diagnosis of these cases. The lack of concordant results between the real-time RT-PCR assays for three serotype O viruses was attributed to four nucleotide mismatches in the 5'UTR PCR primer and probe sites (three substitutions for the sense-primer and one in the TaqMan(®) probe region). Taken together, the laboratory results showed that recent FMD outbreaks that occurred during 2008 in northern and central Sudan were caused by serotypes O and SAT2, while serotype A was last detected in 2006. Phylogenetic analyses of VP1 sequences from these viruses were used to determine the relationships with 23 older viruses from Sudan and other viruses from West and East Africa. For serotype O, closest genetic identities were between concurrent and historical Sudanese isolates, indicating that within-country circulation is an important mechanism by which FMD is maintained year-on-year in Sudan. A similar pattern was also evident for serotype A and SAT2 viruses; however, these lineages also contained recent representative FMD viral isolates from other countries in the region suggesting that long-distance animal movement can also contribute to FMD dispersal across sub-Saharan Africa. These findings provide the first molecular description of FMD viruses that are circulating in Sudan, and highlight that further sampling of representative viruses from the region is required before the complex epidemiology of FMD in sub-Saharan Africa can be fully understood.

Detection of African swine fever virus by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n=38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

Investigations into the cause of foot-and-mouth disease virus seropositive small ruminants in Cyprus during 2007.

In 2007, serological evidence for foot-and-mouth disease (FMD) infection was found as a result of differential diagnostic testing of Cypriot sheep suspected to be infected with bluetongue or contagious ecthyma. Seropositive sheep and goats were subsequently uncovered on ten geographically clustered flocks, while cattle and pigs in neighbouring herds were all seronegative. These antibodies were specific for serotype-O FMD virus, reacting with both structural and non-structural (NS) FMD viral proteins. However, no FMD virus could be recovered from the seropositive flocks. FMD had not been recorded in Cyprus since 1964 and there has been no vaccination programme since 1984. Since all the seropositive animals were at least 3 years old and home-bred, it was concluded that infection had occurred approximately 3 years previously had passed un-noticed and died out spontaneously. It therefore appears that antibodies to FMD virus NS proteins can still be detected around 3 years after infection of small ruminants, but that virus carriers cannot be detected at this time. This unusual situation of finding evidence of historical infection in a FMD-free country caused considerable disruption and alarm and posed questions about the definition of what constitutes a FMD outbreak.

Exanthema in Legionnaires' disease mimicking a severe cutaneous drug reaction.

Legionnaires' disease is an acute bacterial infection, generally caused by Legionella pneumophila, which primarily involves the lower respiratory tract, although it is often associated with multisystemic extrapulmonary features. Cutaneous features are very uncommon and may include erythematous or petechial, macular or maculopapular lesions. We report a male patient who expressed all features of a severe lobular pneumonia. Over the course of the disease the patient developed a livid erythematous, maculopapular exanthem rapidly extending over the entire body. Given the rapid development and target-like appearance of the skin lesions with extensive skin involvement and blister formation, the initial diagnosis was that of a severe cutaneous drug reaction. However, histological examination of biopsy did not confirm this diagnosis, but instead was suspicious for a viral exanthem or a more aggressive inflammatory response due to sensitization to bacterial antigens. L. pneumophila infection was verified during the course of the disease.

Improved diagnosis for nine viral diseases considered as notifiable by the world organization for animal health.

Nine viral diseases included in the World Organization for Animal Health list of notifiable diseases (former list A) were chosen for their contagiousness and high capacity of spreading to improve their diagnosis using new and emerging technologies. All the selected diseases--foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza--are considered as transboundary diseases, which detection causes the prohibition of livestock exportation, and, thus, it leads to high economical losses. The applied diagnostic techniques can fall into two categories: (i) nucleic-acid detection, including padlock probes, real-time PCR with TaqMan, minor groove binding probes and fluorescence energy transfer reaction probes, isothermal amplification like the Cleavase/Invader assay or the loop-mediated amplification technology and the development of rapid kits for 'mobile' PCR and (ii) antigen-antibody detection systems like simplified and more sensitive ELISA tests. Besides, internal controls have been improved for nucleic acid-detecting methods by using an RNA plant virus--Cowpea Mosaic Virus--to ensure the stability of the RNA used as a positive control in diagnostic real-time RT-PCR assays. The development of these diagnosis techniques has required the joint efforts of a European consortium in which nine diagnostic laboratories and an SME who have collaborated since 2004 within the European Union-funded Lab-on-site project. The results obtained are shown in this paper.

Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841.

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

[Results two years after stapler hemorrhoidectomy versus Milligan-Morgan procedure]. / Die Klammernahtresektion bei Hämorrhoiden - eine Bestandsaufnahme nach zweijähriger Anwendung - Vergleich der Ergebnisse mit der Technik nach Milligan-Morgan -

The aim of this investigation was to demonstrate possible advantages of stapler hemorrhoidectomy in comparison to the Milligan-Morgan procedure. 96 patients with an average age of 54 years were treated in a two year period (7/1998-8/2000) by stapler hemorrhoidectomy. The complication rate was 12.5 % and included one mechanical stapler defect, two cases of bleeding, five cases of urinary retention and four of perianal edema. The use of analgesics was small with 70 % requiring no medication at all. Hospitalisation post-operatively was 3.3 days with patients under 65 years old and 4.7 days in those over 65 years. These data were compared retrospectively to that of Milligan-Morgan hemorrhoidectomy (214 patients) performed between January 1990-December 1997. The stapler patients had less pain, fewer complications and shorter hospitalisation. Using a questionnaire, all stapler patients and 50 Milligan-Morgan controls were evaluated. 78 % responded at 13.8 months after stapler hemorrhoidectomy, 63 % 54.1 months after Milligan-Morgan. The degree of satisfaction was high in both groups (93 vs 94 %). One patient in the Milligan-Morgan group suffered a recurrence. No further symptoms had been experienced by 57 % after stapler, 68 % after Milligan-Morgan procedure. Faecal continence represents a problem in the stapler group. Stapler hemorrhoidectomy is an effective treatment for IIIrd degree hemorrhoids. In comparison to the Milligan-Morgan procedure, it has advantages in the early post-operative period. Defecation problems can occur with an unknown prognosis. Without long-term results and because of the comparatively high cost of the procedure the indication for stapler hemorrhoidectomy should be carefully made.

Expression of the human alpha1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis.

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell-based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human alpha1, 2-fucosyltransferase (H-transferase, HT). H-transferase expression modifies the cell surface carbohydrate phenotype of the xenogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galalpha1,3-Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine-inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galalpha1,3-Gal epitope, reduced XNA reactivity, and an increased resistance to human serum-mediated cytolysis. Transgenic pigs that express H-transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.

The use of transgenic animals in biotechnology.

The development of a technique that allows for oocyte and early embryo manipulation is one of the major scientific endeavors in the field of genetic manipulation for animal disease models, basic science in gene regulation and commercial applications. Dr. Ralph Brinster is one of the most prestigious scientists in the development of this science. Through his direction and support, the undertaking of the mechanisms that are involved in the earlier stages of embryology have been productive and enlightening. This paper outlines just some of the experimental successes that evolved from Dr. Brinster's insight and mentorship of one of his pupils. The essay outlines several experimental approaches that have contributed to this field. Specifically, it addresses how the mouse oocyte and the zygote respond to messenger RNA when introduced into the cell, in comparison to comparable non-mammalian species embryos. In addition, this paper discusses some transgenic animal models, both from a basic science point of view and a commercial extension of these techniques. This extension of Dr. Brinster's pioneering work is through technology that allows for the introduction of foreign DNA that can be expressed in targeted organs, such as the mammary gland for production of pharmaceuticals for use in clinical applications.

Novel repeat elements direct rat proenkephalin transcription during spermatogenesis.

The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-tau during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5'-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Glycoprotein B genotype of human cytomegalovirus: distribution in HIV-infected patients.

Glycoprotein B (gB) is involved in cell to cell transmission of human cytomegalovirus (HCMV) and may be a critical factor in tissue tropism and viral pathogenesis. We analyzed the distribution of the four known gB genotypes of HCMV in 99 HIV-positive patients. 29 patients had HCMV retinitis, and 70 patients had asymptomatic HCMV infection. DNA was isolated from blood, urine, and aqueous humor, and gB genotypes were determined by PCR and restriction analysis. Infections with gB type 1 were less frequent in patients with retinitis than in patients with asymptomatic HCMV infection (17% versus 37%; p = 0.05). Furthermore, the gB type was correlated with dissemination of infection. In patients with HCMV detected in only one compartment (blood or urine) the gB type 1 was found more frequently than in patients with HCMV detected in at least two compartments (p = 0.01). The data show that gB genotypes differ in their association with clinical disease, and indicate that the gB genotype may contribute to the course of HCMV infection.

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Mouse half embryos: viability and allocation of cells in the blastocyst.

We have investigated the developmental capacity of mouse embryos in which one blastomere was destroyed by lysis at the 2-cell stage. The allocation of cells to the trophectoderm and inner cell mass (ICM) was documented by differential cell counts on single embryos after 2 days under different culture conditions. Viability and further developmental potential were tested by embryo transfer to foster mothers. The conditions used were: (1) in vitro culture in modified BMOC-2 medium, (2) in vivo oviduct transfer to immature (prepuberal) females, and (3) in vivo oviduct transfer to pseudopregnant females. Half embryos almost always fared less well for all parameters of development than control embryos developing under the same conditions. Lower total cell numbers in half embryos were accounted for by decreases in both ICM and trophectoderm with a disproportionate decrease in ICM in smaller embryos. In both half and control embryos, the growth conditions affected the rate of morphological development, the total cell number, and embryo viability. Unlike the effect of halving embryos, the growth condition effects on total cell number can be accounted for primarily by differences in ICM cell number, with trophectoderm cell number remaining constant. These results provide new information on the ability of the mouse embryo to differentially regulate ICM and trophectoderm cell number under different conditions, and confirm our previous work showing the advantage of short-term development in vivo over short-term in vitro culture (Papaioannou and Ebert [1986] J. Reprod. Fertil. 76:603-608).(ABSTRACT TRUNCATED AT 250 WORDS)