Porphyromonas gingivalis culture supernatants differentially regulate interleukin-1beta and interleukin-18 in human monocytic cells.

Porphyromonas gingivalis is a major bacterial species implicated in chornic periodontitis, a disease characterized by inflammatory destruction of the tooth supporting tissues. Its main virulence factors are lipopolysaccharide (LPS) and gingipains, a group of cysteine proteinases. Interleukin (IL)-18 is a potent pro-inflammatory cytokine with structural similarities to IL-1beta. This study aimed to investigate if P .gingivalis regulates IL-1beta and IL-18 in monocytic cells. Monomac-6 cells were challenged with P. gingivalis culture supernatants. Quantitative real-time PCR and ELISA were used to investigate IL-1beta and IL-18 mRNA expression and protein secretion, respectively. P. gingivalis enhanced IL-1beta and IL-18 mRNA expression, the former being induced earlier, but transiently. IL-18 up-regulation was not affected by P. gingivalis heat-inactivation or chemical inhibition of its gingipains, whereas both treatments resulted in 50% reduction of IL-1beta expression. Purified P. gingivalis LPS enhanced both IL-1beta and IL-18 expression. However, only IL-1beta, but not IL-18, secretion was detected, and was up-regulated by P. gingivalis. In conclusion, although IL-1beta and IL-18 belong to the same cytokine family, their gene expression and secretion are differentially regulated in human monocytic cells in response to P. gingivalis. Therefore, cytokines of the IL-1 family may participate via different pathways in the complex pathogenesis of periodontitis.

Porphyromonas gingivalis stimulates TACE production by T cells.

INTRODUCTION: Tumour necrosis factor-alpha converting enzyme (TACE), also known as ADAM17, is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T-cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. METHODS: P. gingivalis 6-day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell-associated TACE levels were measured by enzyme-linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real-time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat-inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. RESULTS: P. gingivalis challenge resulted in a concentration-dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. CONCLUSION: The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell-bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.

Evolution in techniques of laparoscopic pancreaticoduodenectomy: a decade long experience from a tertiary center.

BACKGROUND: Laparoscopic pancreaticoduodenectomy is a technically demanding surgery performed only at few centers in the world. This article aims to describe the evolution of the technique and summarizes the results in our institute over the years. METHODS: Prospective data of patients undergoing laparoscopic pancreaticoduodenectomy from March 1998 to January 2009 was retrospectively reviewed. RESULTS: There were a total of 75 patients (22 females and 53 males) with a mean age of 62 (range, 28-76) years. Conversion rate was 0%, overall postoperative morbidity was 26.7% and mortality rate was 1. 33%. Pancreatic fistula was seen in 6.67%. The mean operating time was 357 min (range 270-650), and the mean blood loss was 74 ml (range 35-410). The average time to the first bowel movement was 3 days and mean hospital stay was 8.2 days (range 6-42). Resected margins were positive in 2.6% of cases. The mean number of retrieved lymph nodes for the malignant lesions was 14 (range 8-22). CONCLUSION: Laparoscopic pancreaticoduodenectomy can be safely performed by highly skilled laparoscopic surgeons. This technique can achieve adequate margins and follow oncological principles. Randomized comparative trials are needed to establish the superiority of laparoscopy versus open surgery.

Transumbilical flexible endoscopic cholecystectomy in humans: first feasibility study using a hybrid technique.

BACKGROUND: Natural-orifice transluminal endoscopic surgery (NOTES) procedures have been tested using numerous approaches, mainly in animals. In humans, only cholecystectomy has been assessed, using a combined transvaginal and transumbilical approach. We present another variant of a hybrid technique for cholecystectomy, namely the combination of a flexible transumbilical double-channel endoscope and a 3-mm rigid transcutaneous trocar placed in the left hypochondrium for liver retraction. PATIENTS AND METHODS: The procedure was attempted in 10 well-selected young patients (M : F = 4 : 6, mean age 29.5 years). Instruments used through the two working channels of the endoscope were either a grasping forceps or snare for grasping and pulling and a hot-biopsy forceps for cold and hot preparation and dissection. Endoclips were used for cystic duct and artery closure. Postoperative analgesia consisted of one intravenous dose of analgesic, followed by oral administration for one further day. Follow-up visits were scheduled at 7 days, 30 days, 90 days, and 6 months. RESULTS: In 4 of the 10 cases the operation had to be converted to conventional laparoscopic cholecystectomy due to difficulty in dissection (in 2 cases) or uncontrollable hemorrhage (2 cases). The mean operating time was 148 minutes. Of the 6 cases in which the procedure was finished by the new approach, cystic artery bleeding occurred in 1 and was successfully clipped. One further patient had a postoperative cystic duct leak with a bilioma, successfully treated by endoscopic retrograde cholangiopancreatography with stenting. Five of the six patients reported themselves as satisfied at 3- or 6-month follow-up. CONCLUSIONS: So far, our endoscope-based transumbilical cholecystectomy technique has not yielded satisfactory results in humans. Further instrument and accessory improvements may increase both success rate and acceptance. Scarless surgery without the inherent risks of a transluminal approach may then become feasible.

Laparoscopic repair of parahiatal hernias with mesh: a retrospective study.

BACKGROUND: Parahiatal hernias are very rare and distinct entities, the diagnosis of which is never made clinically. Laparoscopic repair has been reported in the literature. We present our experiences with the laparoscopic repair of this uncommon type of hernia. PATIENTS AND METHODS: In our institute, we retrospectively identified a total of eight patients with parahiatal hernias from 1999 to 2007, of which four had primary and four had secondary defects. Laparoscopic crural repair was performed for all of the patients, fundoplication wherever indicated and meshplasty in the cases with large defects. Gastropexy was performed for the patient with volvulus. RESULTS: The male:female ratio was 5:3, with a mean age of 46 years and a mean body mass index (BMI) of 29.3 kg/m2. The mean size of the defects was 18 cm2. The mean blood loss during surgery was 50 ml, the mean operative time was 103.5 min and the mean hospital stay was 4 days. One patient had the recurrence of symptoms 1 month after surgery. There were no conversions, recurrences or mortality. DISCUSSION: Primary parahiatal hernias occur as a result of a congenital weakness and secondary defects follow hiatal surgery. The use of a mesh is advisable for large defects and defects of primary type. Secondary hernias following fundoplication do not need a redo fundoplication, but require an adequate crural repair with mesh. Laparoscopic repair of these uncommon hernias is safe, effective and provides all of the benefits of minimally invasive surgery.

Laparoscopic transperitoneal repair of lumbar incisional hernias: a combined suture and 'double-mesh' technique.

BACKGROUND: Lumbar hernias that occur after surgery are called lumbar incisional hernias. Recently, laparoscopic repair of these hernias has been reported with excellent outcomes. This is a retrospective study of our series of patients with lumbar incisional hernias. PATIENTS AND METHODS: We managed 11 patients with lumbar incisional hernias from 1996-2006. All the patients had undergone either nephrectomy or pyeloplasty in the past. Laparoscopic suturing of the defect and reinforcement with mesh were successfully performed for all the patients. RESULTS: There were more males than females, the age range was 42-65 years, and mean operating time was 120 min; discharge was at 1-2 postoperative days. There was no recurrence or mortality. Three cases had seroma, out of which two required aspiration after 60 days. DISCUSSION: Laparoscopic repair provides all the benefits of minimally invasive surgery, and the principles involved in repair of ventral hernias are applied in lumbar incisional hernias as well. Our technique involved suturing of the defect before placing a mesh over the defect. We theorize that approximating the ends of the muscles restores normal anatomy and results in functional improvement. For the larger hernias, we used two meshes to cover the defect--polypropylene and Parietex, sizes being 15 x 15 cm. CONCLUSION: Laparoscopic repair with prosthetic reinforcement is feasible and effective in the treatment of lumbar incisional hernias. Also, suturing of the defect may provide additional benefits.

Laparoscopic repair of suprapubic incisional hernias: suturing and intraperitoneal composite mesh onlay. A retrospective study.

BACKGROUND: Suprapubic hernias are those defects located 4 cm from the pubic symphysis. Our aim is to highlight the laparoscopic repair of suprapubic incisional hernias. PATIENTS AND METHODS: We retrospectively reviewed 17 patients with suprapubic incisional hernias from 1999 to 2007. A modified technique of laparoscopic intraperitoneal composite mesh onlay was performed for these patients. RESULTS: There were 12 females and 5 males with a mean age of 55.9 years and a mean BMI of 30 kg/m(2). The mean hernia size was 87.5 cm(2), with an average mesh size of 234 cm(2). Mean follow-up was 9 months. Complications were seen in five patients, with an overall recurrence rate of 5.8%. DISCUSSION: Suprapubic hernias are difficult to manage because of the complexity of dissection and their anatomic proximity to bony, vascular and nerve structures. The lower end of the mesh should be fixed to the Cooper's ligament and the pubic bone. Laparoscopic repair of these uncommon hernias is safe, effective and provides all the benefits of minimally invasive surgery.

Laparoscopic transhiatal approach for benign supra-diaphragmatic lesions of the esophagus: a replacement for thoracoscopy?

Benign esophageal lesions are rare conditions and tumors account for about 10% of all esophageal neoplasms. Epiphrenic diverticula occur in the distal esophagus (the lower 10 cm). Currently, thoracotomy/thoracoscopy is the most popular approach for these conditions. We present our experience of 13 patients (1994-2006) with benign supra-diaphragmatic esophageal lesions that we treated with a laparoscopic transhiatal approach. The lesions included in the series were lower esophageal tumors (n = 8) and epiphrenic diverticula (n = 5). Laparoscopic transhiatal stapler excisions of diverticulum and enucleation of tumors were performed for all patients. Intra-operative endoscopy was used in all the procedures. All patients had an uneventful recovery except one with posterior diverticulum, who had an anastomotic leak. He had a prolonged hospital stay and recovered eventually. There was no mortality. Benign lesions of the lower third of the esophagus can be adequately treated through the transhiatal route. This is probably superior to the traditional approaches of thoracotomy/thoracoscopy as it does away with increased morbidity while maintaining adequate access. An endoscopy is of great value in localizing the lesion and assessing the esophageal lumen size during the application of staples. A laparoscopic transhiatal excision is technically feasible for all benign supra-diaphragmatic lesions and epiphrenic diverticula and is the approach of choice.

Paraphimosis revisited: is chronic paraphimosis a predominantly third world condition?

Paraphimosis is an acute condition characterized by all the signs of inflammation. Here we describe a 'chronic' variant of paraphimosis where patients often present many days after the initial injury. The aim of this study is to identify and possibly classify this new variation of paraphimosis - the chronic condition. We studied 68 patients with this condition between 1997 and 2005. In all, the prepuce was fibrous and bunched up behind the glans penis and, in some cases, with ulcerations. All patients were treated by excision of the fibrous flap and circumcision, under local anaesthesia. Patients were discharged on the same day on which the excision was done. Follow-up was on the third, seventh and ninetieth days. Long-term follow-up was possible in only 22 patients. This technique of modified circumcision was effective in treating this condition and we think a new classification of paraphimosis is warranted - acute and chronic.

Laparoscopic orchiectomy for the adult impalpable testis--experiences in a rural teaching hospital.

BACKGROUND: Undescended testis is a relatively common condition in boys. The standard treatment is orchiopexy. In adults, orchiopexy is done only if sufficient length can be mobilized. Otherwise, orchiectomy is ideal as undescended testis predisposes to carcinoma. The incidence of carcinoma increases with age. The aim of this study is to highlight the value of laparoscopy in treatment of impalpable testis and simultaneously repair associated hernias. This is our experience in a rural tertiary hospital. METHODS: In our rural hospital, it is not uncommon to see men aged 30 years or more presenting with unilateral absence of testis/empty hemiscrotum. A total of 35 patients were studied. Ultrasonography of the abdomen was done to localize the exact position of the testis; it was detected in 12 cases. A computed tomography (CT) scan was done in the other 23 cases and was positive in 16. The testis was found in the retroperitoneum (close to the internal inguinal ring) in 12 cases and in the inguinal canal in 23 cases. There were associated hernias in 9 patients. RESULTS: Laparoscopy accurately identified the exact location of the missing testis and resection was also accomplished and associated hernias were repaired laparoscopically. DISCUSSION: In countries like India, the majority of the population is poor and illiterate. By the time a boy or young man with an undescended testis arrives at the hospital, it is already too late to do orchiopexy. Even though most of our patients had no symptoms, orchiectomy had to be done because of the risk of torsion and malignant transformation. By using laparoscopy in these patients, the advantages of minimally invasive surgery can be utilized. CONCLUSIONS: Laparoscopic orchiectomy seems to be advantageous and well received by patients. We preferred the laparoscopic approach for the obvious benefits of less pain, better cosmesis, and early discharge.

Minimally invasive management of achalasia cardia: results from a single center study.

BACKGROUND: Since the performance of the first laparoscopic cardiomyotomy for achalasia cardia in 1991, the popularity of the minimally invasive approach for this troublesome disease has been growing. We present our experience of 226 patients who underwent laparoscopic cardiomyotomy and discuss the relevant issues. METHODS: A retrospective analysis was carried out of 226 patients who have undergone laparoscopic cardiomyotomy since 1993. The preoperative workup, surgical technique, and postoperative management are described. RESULTS: Patients included 146 males and 80 females; average age was 36.4 years (range, 6 to 85). Mean duration of symptoms was 1.4 years. Nearly half of the patients (112) had undergone prior pneumatic dilatation. In 20 patients, myotomy alone was done, 44 patients had a Dor's fundoplication, and 162 had Toupet's fundoplication. The average operating time was 96 minutes. Mean postoperative hospital stay was 2.2 days. Dysphagia was eliminated in 88.9% of the patients with an overall morbidity of 4.4% and nil mortality over a mean follow-up of 4.3 years. CONCLUSION: Laparoscopic cardiomyotomy with Toupet's fundoplication is a safe and effective treatment of achalasia cardia. Dor's fundoplication is done selectively, especially when suspicion is present of mucosal injury.

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A-LPS.

Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)-protoporphyrin IX] or hemin [Fe(III)-protoporphyrin IX-Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin-binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, µ-oxo-bisheme {[Fe(III)PPIX]2 O}, which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of µ-oxo-bisheme on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys-gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α-1, 3-mannosyltransferase) produce non-pigmented colonies. However, only those mutants lacking A-LPS showed reduced hemin-binding when cells in suspension were incubated with hemin. Using native, de-O-phosphorylated and de-lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin-binding to O-polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin-binding to A-PS. We conclude that A-LPS in the outer-membrane of P. gingivalis serves as a scaffold/anchor for the retention of µ-oxo-bisheme on the cell surface and pigmentation is dependent on the presence of A-LPS.

Laparoscopy-assisted truncal vagotomy with antecolic posterior gastrojejunostomy for benign gastric outlet obstruction.

BACKGROUND: The aim of this study is to highlight the role of minimally invasive surgery in the form of laparoscopy-assisted truncal vagotomy (TV) with ante-colic posterior gastrojejunostomy (PGJ) for benign gastric outlet obstruction (GOO). GOO is relatively common in southern India due to various factors. From 1994 to 2004, 762 patients with GOO were operated on (open TV with PGJ) in our center. METHODS: From November 2003 to November 2004, 18 patients with GOO underwent the laparoscopic procedure in our unit. The procedure involves laparoscopic TV followed by the ante-colic PGJ performed extracorporeally through a 3.5-cm transverse incision in the upper abdomen. RESULTS: The advantages of this procedure are that pain, hospital stay, size of wound, incidence of incisional hernia, and postoperative complications are reduced and the patient returns to work earlier. The results are comparable to those of a totally laparoscopic TV with PGJ. CONCLUSION: This procedure is relatively easy to perform because the anastomosis is done extracorporeally, and it is less expensive than the use of endostaplers. Thus, more surgeons should be encouraged to perform laparoscopic TV with PGJ.

Laparoscopic jejuno-jejunostomy for afferent loop stasis following truncal vagotomy with posterior gastro-jejunostomy for pyloric stenosis.

A 47-year-old man presented with epigastric pain relieved by bilious vomiting since one month. He had undergone truncal vagotomy with posterior gastrojejunostomy for benign gastric outlet obstruction 2 years ago. Endoscopy showed distension and stasis in the afferent loop, bile gastritis and esophagitis. Laparoscopic Braun jejunojejunostomy relieved his symptoms.

Glucose isomerase: insights into protein engineering for increased thermostability.

Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.

Kinetics of permeation and metabolism of alpha-tocopherol and alpha-tocopheryl acetate in micro-Yucatan pig sin.

The objective of this research was to investigate the permeation and metabolism of alpha-tocopheryl acetate (alpha-TAc) and alpha-tocopherol (alpha-T) from solution and emulsion formulations and to delineate the kinetics of such metabolism. Simple formulations containing alpha-TAc and alpha-T were applied to fresh, viable micro-Yucatan skin dermatomed to a thickness of 250-300 microns, as a finite dose in a flow-through diffusion system. The experiments were stopped at time intervals of 2, 6, 12, and 24 hours. At the end of each time interval, the amounts removed by washing, retained in the stratum corneum (SC), and penetrated into the viable skin and receptor were determined by a validated HPLC method. Receptor concentrations were below the limit of detection. alpha-TAc underwent metabolism in pig skin to the active antioxidant alpha-T. The metabolite appeared as early as two hours after application. The extent of metabolism was highest at 6-12 hours after application. No metabolism was detected in the stratum corneum. Delivery of alpha-T from isopropyl myristate (IPM) solution was more efficient than utilization of alpha-TAc from the same solution. Approximately 1.5% of alpha-T yielded the same viable skin concentration as 5% alpha-TAc. Topical application of alpha-tocopherol or its prodrug acetate was capable of enhancing the overall antioxidant capacity of pig skin. The hydrolytic pathway of alpha-TAc leading to the active antioxidant alpha-T could possibly be saturable.

Effect of formulation on the delivery and metabolism of alpha-tocopheryl acetate.

The effect of delivery system on the permeation and metabolism of alpha-tocopheryl acetate (alpha-TAc) was studied in micro-Yucatan pig skin, which closely resembles human skin. Various alpha-tocopheryl acetate formulations, including a simple isopropyl myristate (IPM) solution, an o/w emulsion, microemulsions, which differed in their oily phase content, and alcoholic and hydroalcoholic gels were made. A suitable HPLC method was developed and validated to separate and quantify alpha-TAc and alpha-tocopherol (alpha-T). Dulbecco's modified phosphate-buffered saline with 3% bovine serum albumin (DMPBS-BSA 3%) served as the receptor media to ensure tissue viability and to maintain skin conditions. Finite doses (5 microl) of the formulations were applied to viable pig skin using a statistically approved randomized complete block design. Data were analyzed using Tukey's studentized range test, and interday variability was estimated using an F-test. About 70% of the active was recovered from the wash, representing the amount adhering to the surface of the skin. alpha-TAc underwent metabolism in pig skin to the active antioxidant, alpha-T. The identity of the HPLC peaks were confirmed by spiking studies using known standards. The extent of metabolism was found to be formulation-dependent. No alpha-T was, however, detected in the stratum corneum. A higher extent of metabolism was obtained for the IPM solution, a microemulsion containing IPM as the oily phase, and the hydroalcoholic gel, when calculated based on the percent of total alpha-TAc permeated in the viable skin. Metabolism occurred in pig skin to the extent of 15-20% in terms of the total amount of alpha-TAc permeated in the viable skin and stratum corneum. Thus the topical delivery and metabolism of alpha-TAc were found to be dependent on formulation.