Characterization of the outer membrane subproteome of the virulent strain Salmonella Typhimurium SL1344.

Outer membrane proteins (OMPs) play an important role in the interaction of bacterial pathogens with host cells. Indeed, some OMPs from different Gram-negative bacteria have been recognized as important virulence factors for host immune recognition. This scenario has led to the study of the outer membrane (OM) subproteome of pathogenic bacteria as an essential step for gaining insight into the mechanisms of pathogenesis and for the identification of virulence factors. Although progress in the characterization of the OM has recently been reported, detailed protein composition of this subcellular localization has not been clearly defined for most pathogens. Salmonella enterica serovar Typhimurium is not only a leading cause of human gastroenteritis in high-income countries but is also one of the main causes of invasive non-typhoidal salmonellosis (iNTS) in middle- and low-income countries. The incidence of non-typhoidal salmonellosis is increasing worldwide, causing millions of infections and deaths among humans each year. Regrettably, antimicrobial resistance to a broad spectrum of antibiotics is common among non-Typhi Salmonella strains. Therefore, the development of vaccines targeting this leading invasive pathogen is warranted. In the present study we have identified the OM protein profile of the virulent S. Typhimurium strain SL1344 by means of sarkosyl extraction. SIGNIFICANCE: Salmonella enterica serovar Typhimurium causes food-borne gastroenteritis around the world, but is also responsible for a more serious manifestation of the disease through a form of invasive illness, invasive non-typhoidal Salmonella (iNTS) disease, which is considered a major public health problem in low- and middle-income countries. Even though some studies have been carried out in order to characterize the outer membrane subproteome of this human pathogen, as far as we know, this is the first report in which the most indicated methodology has been used in order to extract the outer membrane proteins and to check the presence of the proteins in the SL1344 genome; indeed all the previous studies were carried out before the genome sequence was available in 2012. Outer membrane proteins are key elements for the interaction of Gram-negative bacteria with their environment ­ including the host ­ and have fundamental roles in both infection and resistance processes. Therefore, a detailed knowledge of the outer membrane composition will certainly play a key role in providing new targets to fight this pathogen in further studies.

Extended spectrum ß-lactamase-producing Escherichia coli faecal carriage in Spanish travellers returning from tropical and subtropical countries.

The aim of this study was to investigate the prevalence of extended-spectrum ß-lactamase (ESBL) -producing Escherichia coli in stool samples from 457 patients with travellers' diarrhoea who had travelled to tropical and subtropical countries. Ninety-seven ESBL-producing E. coli strains were isolated from 17.9% of the patients (82/457). CTX-M-15 was the most prevalent enzyme (80%) and India was the most visited country and showed the highest prevalence of positive samples (37.4%).

Four-dimensional sonographic evaluation of avulsion of the levator ani according to delivery mode.

OBJECTIVE: To determine the frequency of avulsion of the levator ani muscle in primiparous women according to delivery mode, using introital four-dimensional ultrasonography. METHODS: We performed a prospective observational study at a tertiary obstetric unit. One hundred and eighty primiparous women were included and divided into three groups: normal vaginal delivery without episiotomy, forceps delivery and Cesarean section groups. Between 40 and 120 days after delivery, four-dimensional ultrasonography was performed in order to evaluate the integrity of the levator ani muscle. The operator was blinded to all clinical data and was not aware of delivery mode. The influence of other variables associated with delivery such as birth weight, body mass index, maternal age and use of epidural anesthesia was also studied. RESULTS: Avulsion of the puborectalis component of the levator ani muscle was detected on ultrasonography in 61.7% of women who had undergone a forceps delivery, compared with 13.3% of those who had had a normal vaginal delivery and 0% of those who had had a Cesarean section. Bilateral avulsion was observed in 12/60 (20.0%) of the forceps group and in 2/60 (3.3%) of the normal vaginal delivery group (P < 0.001). Other variables did not seem to influence prevalence. CONCLUSIONS: Forceps delivery is associated with an increased rate of avulsion of the puborectalis component of the levator ani muscle. The effect of forceps use is independent of other delivery-related variables.

The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability.

In a previous study we reported that the immunolabelling of GLUT3, HSP90AA1, and Cu/ZnSOD proteins on boar sperm did not show differences between good and poor freezability ejaculates, in terms of a qualitative analysis based on location and reactivity of these proteins at 17 degrees C and at 240 min post-thaw. Since predicting the ejaculate freezability is considerably important in sperm cryopreservation procedures, the objective of the present study was to quantify the expression of these three proteins in good and poor freezability ejaculates. For this purpose, 10 ejaculates from 9 Piétrain boars were cryopreserved and their sperm quality assessed in the three main steps of the freezing process (17 degrees C, 5 degrees C, and 240 min post-thaw). After this assessment, the 10 ejaculates were clustered for freezability on the basis of their sperm progressive motility and membrane integrity at 240 min post-thaw. From the whole ejaculates, only four good and four poor freezability ejaculates displaying the most divergent values were selected for a western blot assay using sperm samples coming from the three mentioned freezing steps. Protein levels through densitometry were significantly different between good and poor freezability ejaculates for Cu/ZnSOD at 240 min post-thaw (P

Effects of matrix filtration of low-quality boar semen doses on sperm quality.

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 +/- 0.5 cm), CM Sephadex (length 5 +/- 0.5 cm), glass wool (length 2 +/- 0.5 cm) or glass bead (length 10 +/- 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca((R)) 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l-lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l-lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.

A sensory neuron subpopulation with unique sequential survival dependence on nerve growth factor and basic fibroblast growth factor during development.

We characterized a subpopulation of dorsal root ganglion (DRG) sensory neurons that were previously identified as preferential targets of enkephalins. This group, termed P-neurons after their "pear" shape, sequentially required nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) for survival in vitro during different developmental stages. Embryonic P-neurons required NGF, but not bFGF. NGF continued to promote their survival, although less potently, up to postnatal day 2 (P2). Conversely, at P5, they needed bFGF but not NGF, with either factor having similar effects at P2. This trophic switch was unique to that DRG neuronal group. In addition, neither neurotrophin-3 (NT-3) nor brain-derived neurotrophic factor influenced their survival during embryonic and postnatal stages, respectively. The expression of NGF (Trk-A) and bFGF (flg) receptors paralleled the switch in trophic requirement. No single P-neuron appeared to coexpress both Trk-A and flg. In contrast, all of them coexpressed flg and substance P, providing a specific marker of these cells. Immunosuppression of bFGF in newborn animals greatly reduced their number, suggesting that the factor was required in vivo. bFGF was present in the DRG and spinal cord, as well as in skeletal muscle, the peripheral projection site of P-neurons, as revealed by tracer DiIC(18)3. The lack of requirement of NT-3 for survival and immunoreactivity for the neurofilament of 200 kDa distinguished them from muscle proprioceptors, suggesting that they are likely to be unmyelinated muscle fibers. Collectively, their properties indicate that P-neurons constitute a distinct subpopulation of sensory neurons for which the function may be modulated by enkephalins.

Embolization of a mesenteric arteriovenous fistula following pancreatic allograft: the steal effect.

The first pancreatic transplant was performed in 1966 by Kelly and Lillehei at the University of Minnesota (1, 2). Nearly 30 years later, Ozaki et al., published the first occurrence of an arteriovenous fistula (AVF) in a transplanted allograft bundle (3). From its early days as a radical and experimental procedure in the treatment of insulin-dependent diabetes mellitus, this operation has progressed to become routine in many major medical centers. However, the incidence of technical complications remains relatively high, ranging from 10 to 40% (1). The list of potential complications includes allograft pancreatitis, vascular thrombosis, hemorrhage, pseudoaneurysm formation, anastomotic leaks, intra-abdominal infections, and, al. though rare, AVF. Lowell et al., in 1993, reported this last complication in 3 of 90 consecutive pancreatic recipients (4). These same authors promoted the theory that AVF formation was directly related to procurement technique: a nonspecific "blind ligation" of mesenteric vessels along the inferior pancreatic border. However, this approach continues to be used commonly. We have identified the occurrence of an allograft superior mesenteric artery-superior mesenteric vein (SMA-SMV) AVF in a pancreas-after-kidney (PAK) transplant recipient.

An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo.

Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.

Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection.

Although recombinant adenoviruses are attractive vectors for gene transfer to airway epithelia, they have proven to be relatively inefficient. To investigate the mechanisms of adenovirus-mediated gene transfer to airway epithelia, we examined the role of adenovirus fiber and penton base, the two proteins involved in attachment to and entry of virus into the cell. We used human airway epithelia grown under conditions that allow differentiation and development of a ciliated apical surface that closely resembles the in vivo condition. We found that addition of fiber protein inhibited virus binding and vector-mediated gene transfer to immature airway epithelia, as well as to primary cultures of rat hepatocytes and HeLa cells. However, fiber protein had no effect on vector binding and gene transfer to ciliated airway epithelia. We obtained similar results with addition of penton base protein: the protein inhibited gene transfer to immature epithelia, whereas there was no effect with ciliated epithelia. Moreover, infection was not attenuated with an adenovirus containing a mutation in penton base that prevents the interaction with cell surface integrins. These data suggest that the receptors required for efficient infection by adenovirus are either not present or not available on the apical surface of ciliated human airway epithelia. The results explain the reason for inefficient gene transfer and suggest approaches for improvement.

Cationic lipid-mediated transfer of the hIL-10 gene prolongs survival of allogeneic hepatocytes in Nagase analbuminemic rats.

Gene transfer techniques can be used as a drug delivery system to achieve local immunosuppression. We performed a series of experiments to identify the cationic lipid that most efficiently transfects isolated, cultured, rat hepatocytes; to optimize conditions for efficient transfection; to determine the duration of gene expression in vitro; and finally, to determine the survival of allogeneic hepatocytes transplanted into Nagase rats. Our results suggest that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. In addition, the following conditions appear to optimize transfection efficiency: a DNA:DOTAP ratio of 1:6; a 24 exposure time of the hepatocytes to the DNA-DOTAP complex; a DNA dose of 4 microg/35 mm culture plate seeded with 2.5x10(5) rat hepatocytes. When transfected as described above, cultured hepatocytes expressed the hIL-10 gene for approximately 14 days. Accordingly, Nagase rats transplanted with 4x10(7) DOTAP-hIL-10 transfected, allogeneic hepatocytes had an abrupt rise in serum albumin levels that peaked within 7 days of the transplant, decreased abruptly after 15 days, and approached baseline by day 40. In contrast, control animals had a smaller albumin peak that returned to baseline within 10 days (P<0.01). In all animals, serum hIL-10 levels were undetectable when tested. We conclude that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. Furthermore, hIL-10 transfected hepatocytes have a prolonged survival in an allogeneic host which is probably limited by loss of gene expression. Further studies using other vectors capable of prolonged gene expression will help determine if indefinite hIL-10 gene expression leads to indefinite graft survival.

Gastrointestinal problems in the immunocompromised host. A review for surgeons.

As the immunocompromised patient population grows, the gastrointestinal surgeon is increasingly called upon to make complex diagnostic and therapeutic decisions. The surgeon should first identify the patient as immunocompromised and then categorize the probable degree of immunocompromise as mild, moderate, or severe. Mildly immunocompromised patients tend to present late and with minimal symptoms, but the disease entities are the same ones seen in the general population. Moderately and severely immunocompromised patients may also develop the usual surgical problems, but the differential diagnosis is expanded to include complications of the immunocompromised state or complications of the underlying problem which caused the immune compromise. The expanded differential diagnosis includes infections with atypical organisms, opportunistic neoplasms, neutropenic enterocolitis, complications of medications, and forms of biliary tract disease not seen in the general population. Advances in oncology, transplantation, and the treatment of AIDS, have extended the life expectancy of these patients and increased the immunocompromised population. Prompt appropriate operative therapy may be lifesaving when surgical complications develop.

Circadian variation of tacrolimus disposition in liver allograft recipients.

The aim of this study was to characterize the circadian variation of oral tacrolimus disposition in 8 stable liver allograft recipients. In the steady state, a total of 23 blood samples was taken before and after tacrolimus administration during a 24-hr period and the pharmacokinetic parameters were compared. The area under the curve (AUC) of tacrolimus after the morning dose was significantly larger than after the evening dose (211+/-43 ng x hr/ml [morning] vs. 179+/-45 ng x hr/ml [evening], P=0.02). The time to peak (Tmax) was significantly shorter after the morning dose than after the evening dose (1.6+/-0.7 hr [morning] vs. 2.9+/-0.6 hr [evening], P=0.002). The peak (Cmax) was significantly higher after the morning dose than after the evening dose (32.2+/-10.2 ng/ml [morning] vs. 19.1+/-4.3 ng/ml [evening], P=0.003). However, the trough (Cmin) was not significantly different between the morning dose and the evening dose (13.1+/-3.9 ng/ml [morning] vs. 13.3+/-4.4 ng/ml [evening], P=0.4). This study demonstrated that tacrolimus disposition in liver transplant patients was determined by administration time.

Intrahepatic arterial chemoembolization for hepatocellular carcinoma and metastatic neuroendocrine tumors in the era of liver transplantation.

Surgical resection has been the standard approach for primary and metastatic liver tumors. Long-term survival, however, is limited because of recurrence or hepatic decompensation. Failure of chemotherapeutic regimens or liver transplantation (OLT) to prevent recurrence has resulted in the need for multimodality therapies. We report our experience with preoperative hepatic arterial chemoembolization (CET) followed by OLT in highly select patients. Over a 33-month period, 23 of 41 patients (56%) referred with primary (n = 16) or metastatic neuroendocrine (n = 7) liver tumors met eligibility requirements. Despite mild, self-limited chemical hepatitis, CET was well tolerated in all but three elderly patients who succumbed to liver failure. Four of five patients ultimately received OLT. Three are alive and free of disease at a mean followup of 17 months, one died of recurrent hepatocellular carcinoma, and one (NET) remains well at 33 months with elevated glucagon levels but no measurable disease. All NET patients are alive with resolution of hormonal symptoms. Four of five noncirrhotic patients died of disease, and one has progressive tumor growth. Although OLT following CET achieves superior survival, its application is limited to a minority of patients with such tumors. Careful pretreatment staging and patient selection combined with caution in the use of CET in elderly cirrhotic patients is critical to the success of such therapies.

Antibody-mediated hemolytic anemia following ABO-mismatched organ transplantation: contributory role of HLA matching and polyclonal antilymphocyte globulin.

We report three instances of antibody-mediated hemolytic anemia following ABO-mismatched, but compatible, renal (n = 2) and simultaneous pancreas-kidney (n = 1) transplantation. The two renal allograft recipients had received a 6-antigen matched transplant; one received polyclonal antilymphocyte globulin to treat an early rejection episode. The simultaneous pancreas-kidney transplant received polyclonal antilymphocyte globulin (ALG) as part of a quadruple therapy induction regimen. All three patients developed severe, but self-limited, antibody-mediated hemolytic anemia within two weeks of their transplants. Serologic testing demonstrated the hemolysis to be antibody mediated; furthermore, testing of the ALG lots demonstrated high titers of anti-red blood cell antibodies. The possible contribution of ALG and HLA matching to the hemolysis seen in these patients after ABO mismatched organ transplantation is discussed.