Pre-operative fluid fasting for adult elective surgery.
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Adult↗
Biomedical subjects
P J Desai
Publications and source records attributed to P J Desai.
Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells.
In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.
Cell Line, Transformed↗
Tissue removal utilizing Steiner Morcellator within a LapSac: effects of a fluid-filled environment.
BACKGROUND AND PURPOSE: Tissue removal can be a simple process of withdrawal of the entire organ, piecemeal removal with surgical clamps, or mechanical morcellation. Different mechanical morcellators exist that each have advantages and disadvantages. We have investigated a particular morcellator having an internal mechanized blade system that increases the chances of damage to tissue isolation sacks but removes large volumes of intact organ that can more readily be evaluated histologically. The primary premise of this investigation is that a fluid-filled sack would be less likely to be damaged by the activated blades of the morcellator. MATERIALS AND METHODS: Utilizing a Steiner Morcellator (Karl Storz, Culver City, CA), two porcine kidneys were morcellated within the large LapSac (Cook Urological, Spencer, IN). Two environmental variables were evaluated: dry sac morcellation and fluid-filled sac morcellation. Each session was timed, fluid leakage identified, grasping of the sacks quantified, and gross spillage noted. The tissues were submitted for pathologic evaluation to quantify any differences grossly or histologically. All LapSacs were inspected for gross violation and inflated to distention with fluid to check for tiny leaks. RESULTS: The Steiner Morcellator worked much better within the confines of the LapSac filled with fluid. There were no perforations in our experimental setting. It was not possible discern use of fluid-filled sacks histologically. CONCLUSIONS: The Steiner Morcellator can be utilized safely in the LapSac if cautious observation and fluid-filled sack conditions are maintained. The extracted tissue is easily evaluated histologically.
Animals↗
Running suturing: the ideal length facilitates this task.
BACKGROUND AND PURPOSE: Laparoscopic intracorporeal suturing is difficult, a complex task involving several integrated skills such as needle handling, suturing, and knotting. Running suturing is even more complex in the closed environment secondary to the angles of the suture lines, the tension maintained on the suture line, and the need to secure the ends, including tying a knot from the tail of the suture to the loop of the preceding stitch. We have hypothesized that the ideal suture length facilitates this process, and this study was specifically designed to determine the ideal suture length for an intracorporeal running suture. MATERIALS AND METHODS: Latex phantoms were incised for 1 or 2 cm, and each was marked with black points to indicate suture entrance and exit sites. These marks were placed 2 mm from the incision, starting 2 mm proximally and ending 2 mm distally. Suture lengths could then be varied in direct proportion to the length of the incision, keeping all of these points as constants. The ratios of suture length:incision length were 9:1, 10:1, and 11:1. One surgeon performed all of the running suturing utilizing dry 3-0 coated polyglactin with a tapered SH needle. The times needed to create running suture lines were recorded (seconds), the number of ideal entrance and exit points tabulated, and the number of technical errors (missed movements resulting in prolonged suturing) recorded. RESULTS: The lengths of both the incision and the suture affect the ability to perform laparoscopic intracorporeal running suturing. As the incision increased, the suture:incision ratio of 9:1 resulted in the most efficient results (quickest suturing and fewest errors). In smaller suture lines (1 cm), the ratios 10:1 and 11:1 appeared better. CONCLUSIONS: A suture:incision ratio of 9:1 is best for longer suture lines and 10:1 is better for short suture lines.
Evaluation Studies as Topic↗
Laparoscopic augmentation cystoplasty using the novel biomaterial Surgisis: small-intestinal submucosa.
BACKGROUND AND PURPOSE: Urinary bladder augmentation is indicated for diverse conditions, including neurogenic bladder, cancer resection, spinal cord injury, and congenital anomalies. The ideal cystoplasty material is yet to be described. Native gastrointestinal segments commonly used are limited by leakage and small-bowel obstruction, metabolic/nutritional abnormalities, calculi, and malignancy. This study assessed laparoscopic bladder augmentation with porcine small intestinal submucosa (SIS). MATERIALS AND METHODS: Five female pigs (<25 kg) were prepared for surgery under general anesthesia. After Veress needle insufflation, a main 10-mm trocar was placed in the midline for the laparoscope, with two lateral 10-mm ports added for operative instruments. The bladder dome was incised, and a patch of SIS was sewn into the bladder using running 2-0 Vicryl. Three animals served as technical studies. Two additional sows underwent long-term survival surgery: one undiverted and one diverted via a Stamey suprapubic catheter. RESULTS: There were no operative losses. The mean operative time was 140 minutes. The SIS graft held the sutures without tearing. Laparoscopic survey revealed no urine leaks at bladder closure. All five animals voided postoperatively. Urinary extravasation was evident in the three undiverted technique animals. In the other two sows, cystoscopy at 7 days showed intact suture lines without evidence of urinary extravasation and with normal vesicular volumes. Tissue growth was evident, but the graft margins were still discernible. CONCLUSIONS: Laparoscopic bladder augmentation was possible using SIS but at minimal volumes. There were no operative complications; however, the material was difficult to deploy and may benefit from application of an absorbable scaffold. Postoperative urinary drainage is necessary. Further studies will optimize the graft configuration for maximal augmentation.
Animals↗
Pathogenic neisseriae can use hemoglobin, transferrin, and lactoferrin independently of the tonB locus.
Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis and Neisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb(+) variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb(+) variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the Neisseria Hb(+) variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described tonB locus.
Bacterial Outer Membrane Proteins↗
A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells.
The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KDeltaUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KDeltaUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KDeltaUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KDeltaUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KDeltaUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.
Capsid↗
Iron acquisition in the pathogenic Neisseria.
Pathogenic Neisseria species need to obtain iron from the host to grow in vivo. Several iron-transport systems are known, and regulation of Neisseria iron-transport genes occurs via the transcriptional regulator Fur. There is evidence that the ability to transport iron is crucial to the survival of these organisms in vivo.
Animals↗
Analysis of Fur binding to operator sequences within the Neisseria gonorrhoeae fbpA promoter.
The gene encoding Neisseria gonorrhoeae periplasmic binding protein FbpA contains two regions whose sequences exhibit homology with the Escherichia coli ferric uptake regulator protein (Fur) consensus binding sequence. In this study, DNase I footprinting experiments were employed to characterize the operator sequences within the fbpA promoter region to which E. coli Fur binds. A 160-bp fragment encompassing the promotor region and the putative iron boxes of the fbpA promoter was incubated with Fur, DNaseI was added, and the products of these reactions were sequenced to identify nucleotide peaks that were protected. At 50 nM Fur, a protected region that spanned 33 bp and extended 19 bp upstream and 8 bp downstream of the -35 region of the fbpA promoter was observed. At higher concentrations of Fur (75 and 100 nM), an extension of this protected region upstream of the -35 region was observed. Introduction of a plasmid carrying an fbpA-cat transcriptional fusion in E. coli H1717 (Fur+) resulted in an 88% induction of chloramphenicol acetyltransferase expression under conditions of iron restriction; however, chloramphenicol acetyltransferase expression was not responsive to iron in E. coli H1745 (Fur-), indicating that transcriptional regulation of fbpA in response to iron occurs via the negative regulator Fur. The extent of the fbpA operator sequence (42 bp), as defined by our footprinting analysis, would suggest the binding of two Fur repressor dimers.
Bacterial Outer Membrane Proteins↗
Binding and accumulation of hemin in Neisseria gonorrhoeae.
The ability to utilize hemin and hemin-containing compounds for nutritional iron (Fe) uptake has been documented for several pathogenic bacteria. Neisseria gonorrhoeae can utilize free hemin as a source of Fe for growth; however, little is known concerning the mechanisms involved in hemin transport. In this study we have characterized the binding and accumulation of hemin by N. gonorrhoeae and defined the specificity of the gonococcal hemin receptor. N. gonorrhoeae F62 was grown in a chemically defined medium containing the iron chelator Desferal, and hemin transport was initiated by the addition of [59Fe]hemin (4.0 or 8.0 microM; specific activity, 7.0 Ci/mol). 59Fe uptake from radiolabeled hemin by N. gonorrhoeae was energy dependent, and 59Fe was shown to accumulate in the cell at a constant rate during logarithmic growth. However, we observed a decrease in the uptake of 59Fe from radiolabeled hemin when inorganic iron was present in the growth medium. Binding of 59Fe from radiolabeled hemin was inhibited by the addition of either cold hemin, hematoporphyrin, or hemoglobin, but not by ferric citrate. Although [14C]hemin was found to support the growth of N. gonorrhoeae, we did not detect the uptake of 14C from radiolabeled hemin. Extraction of the gonococcal periplasmic ferric binding protein (Fbp) from cultures grown with [59Fe]hemin indicated that a majority of the 59Fe was associated with the Fbp. Taken together, the results presented here indicate that hemin binds to a gonococcal outer membrane receptor through the protoporphyrin portion of the molecule and that following binding, iron is removed and transported into the cell, where it is associated with the gonococcal periplasmic ferric binding protein, Fbp.
Binding, Competitive↗
Excretion of non-infectious virus particles lacking glycoprotein H by a temperature-sensitive mutant of herpes simplex virus type 1: evidence that gH is essential for virion infectivity.
A temperature-sensitive mutant of herpes simplex virus type 1, tsQ26, was shown to contain an amino acid substitution in glycoprotein H (gH). The mutant entered cells efficiently at the non-permissive temperature and replicated to give nearly normal yields of intracellular infectivity. The intracellular virions contained, predominantly, an immature form of gH and no gH was found on the surface of infected cells. Excreted virions were devoid of gH and were not infectious. Virions excreted at the permissive temperature were infectious and contained gH and no loss of gH resulted from incubation of these virions at the non-permissive temperature. The temperature-sensitive phenotype apparently results from the loss of gH from virions during their transport to the cell surface, and since loss of gH is accompanied by loss of infectivity we conclude that gH is an essential component of the infectious virion.
Animals↗
Penicillinase-producing Neisseria gonorrhoeae in Ndola, Zambia.
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Female↗