Salmonella enterica Typhimurium engineered for nontoxic systemic colonization of autochthonous tumors.

Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium (S. Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S. Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 106 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.

Virulence-attenuated Salmonella engineered to secrete immunomodulators reduce tumour growth and increase survival in an autochthonous mouse model of breast cancer.

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

Standardized irrigation technique reduces intraabdominal abscess after appendectomy.

PURPOSE: The utility of irrigation at the time of appendectomy for acute appendicitis has been debated, with recent studies showing no benefit to irrigation. In our practice, two techniques have been used; one in which irrigation was at the discretion of the surgeon, and one in which irrigation was standardized. The standardized irrigation technique involved large volume (3-12 l) irrigation in small, focused, directed aliquots to achieve optimal dilution. We sought to retrospectively assess whether the standardized large volume irrigation technique was associated with measurably reduced intraabdominal infection. We hypothesized that there would be no difference in intraabdominal infection rate. METHODS: Medical records for cases of appendectomies performed for acute appendicitis, years 2007 through 2017, were reviewed (n = 432). Rate of subsequent abdominal infection was compared between patients who underwent the standardized large volume irrigation technique compared to those who did not using Fisher's exact test; p < 0.05 was considered significant. RESULTS: For patients that underwent the standardized large volume irrigation technique there were no (0/140) subsequent abdominal infections within the study period, compared with a rate of 6.2% (18/292) for all other patients (p value 0.001). Among cases that had a perforated appendix (n = 105), the rates were 0% (0/31) compared to 18.9% (14/74; p value 0.009). CONCLUSIONS: Utilization of a standardized large volume irrigation technique with the objective of serial dilution is associated with a significantly lower rate of subsequent abdominal infection, even among cases with a perforated appendix. Prospective studies are needed to evaluate this technique. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Treatment study.

Combined laparoscopic-fluoroscopic technique for primary gastrojejunostomy button tube placement.

BACKGROUND: Gastrojejunostomy (GJ) tubes are frequently used to provide pediatric enteral nutritional support for pediatric patients. Various placement methods have been described, each with attendant advantages and disadvantages. DESCRIPTION OF THE OPERATIVE TECHNIQUE: We present a technique for primary laparoscopic/fluoroscopic GJ button tube placement designed to avoid delay in placement of the jejunal limb, and difficulties associated with endoscopic-assisted and primary fluoroscopic placement. RESULTS: There were 52 gastrojejunostomy button tubes placed via this technique in patients ranging from 3.8 to 90.3 kg in weight. Three postoperative complications were identified; one bowel perforation on postoperative day two, and two tube dislodgements within 30 days. CONCLUSION: The described technique was uniformly effective and was associated with a low complication rate (5.8%).

Peritoneal dialysis catheter placement, outcomes and complications.

PURPOSE: Peritoneal dialysis (PD) is a commonly used method for renal support in pediatric patients and can be associated with the risk of post-surgical complications. We evaluated method of placement of PD catheters with regard to post-surgical complications. METHODS: PD catheters placed at two institutions between 2005 and 2017 were reviewed. Complication rates were evaluated based on method of placement, delayed usage, omentectomy, and patient age using Fisher's exact test, two-sided, with significance set at 0.05. Factors influencing complication were evaluated with multivariate logistic regression and Kaplan-Meier survival analysis. RESULTS: There were 130 patients with 157 catheters placed, ranging in age from 1 day to 23 years. There was no significant difference in complication rate by method of placement or delayed usage. Infants were significantly more likely to experience leakage (21% vs 8%, p 0.036) and hernias (15% vs 5%, p 0.030). Patients that underwent an omentectomy were less likely to require a catheter replacement (7% vs 27%, p 0.004), and the catheters had a significantly higher survival rate (p 0.009). We found that laparoscopic intervention resulted in catheter salvage. Lateral exit sites may be a risk factor for catheter migration in some patients. CONCLUSIONS: Omentectomy is associated with longer PD catheter survival. Laparoscopic salvage of dysfunctional catheters may be a valuable adjunct in management.

White paper on microbial anti-cancer therapy and prevention.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

Necrotizing Enterocolitis in Two Siblings and an Unrelated Infant with Overlapping Chromosome 6q25 Deletions.

The pathogenesis of necrotizing enterocolitis (NEC) remains poorly understood but is thought to be multifactorial. There are no specific recurring chromosomal abnormalities previously associated with NEC. We report 3 cases of intestinal necrosis associated with large chromosome 6 deletions. The first patient was found to have a 7.9-Mb deletion of chromosome 6 encompassing over 40 genes, arr[GRCh37] 6q25.3q26(155699183_163554531)×1. The second patient had a 19.5-Mb deletion of chromosome 6 generated by an unbalanced translocation with chromosome 18, 46,XY,der(6)t (6;18)(q25.1;p11.23), arr[GRCh37] 6q25.1q27(151639526_ 171115067)×1, 18p11.32p11.23(131700_7694199)×3, which included the whole 7.9-Mb region deleted in the first patient. The third patient was the younger sibling of the second patient with an identical derivative chromosome 6. The shared abnormal chromosome 6 region includes multiple genes of interest, particularly EZR. Mouse models have demonstrated that Ezr is expressed in microvillar epithelium and helps regulate cell-cell adhesion in the gut. We hypothesize that deletion of this shared region of 6q leads to gastrointestinal vulnerability which may predispose patients to intestinal necrosis.

3D Printed Organ Models with Physical Properties of Tissue and Integrated Sensors.

The design and development of novel methodologies and customized materials to fabricate patient-specific 3D printed organ models with integrated sensing capabilities could yield advances in smart surgical aids for preoperative planning and rehearsal. Here, we demonstrate 3D printed prostate models with physical properties of tissue and integrated soft electronic sensors using custom-formulated polymeric inks. The models show high quantitative fidelity in static and dynamic mechanical properties, optical characteristics, and anatomical geometries to patient tissues and organs. The models offer tissue-mimicking tactile sensation and behavior and thus can be used for the prediction of organ physical behavior under deformation. The prediction results show good agreement with values obtained from simulations. The models also allow the application of surgical and diagnostic tools to their surface and inner channels. Finally, via the conformal integration of 3D printed soft electronic sensors, pressure applied to the models with surgical tools can be quantitatively measured.

Radial umbilical dermatofasciolysis to invert the skin following umbilical herniorrhaphy.

BACKGROUND: Umbilical hernia is a common congenital anomaly, and can result in the appearance of a protuberant umbilicus. In select cases, inversion of the umbilical skin can be impaired by the presence of thickened dermis or fascial remnants of the umbilical stalk. DESCRIPTION OF OPERATIVE TECHNIQUE: After umbilical herniorrhaphy, the skin is everted over the left index finger and radial partial thickness incisions in the fascia and dermis of the undersurface of the umbilicus. The umbilical skin is then inverted and secured to the fascia. CONCLUSION: This operative technique can allow complete inversion of the umbilical skin creating an aesthetically appealing umbilical hernia repair.

Intraoperative temperature regulation in children using a liquid-warming garment.

PURPOSE: Children undergoing operative intervention while induced under general anesthesia are at risk for experiencing a significant decrease in core body temperature that can lead to adverse systemic effects. Given that the head contributes an estimated 18% of a child's body surface area, we theorized that a liquid-warming garment applied to the head could control a pediatric patient's core body temperature during surgical procedures. METHODS: Patients undergoing elective, non-cranial, general surgical procedures were enrolled in the study. A head garment with an embedded network of tubing was placed on the patient. The garment connected to a computer-controlled water bath that managed the temperature of the water in the tubing through a feedback mechanism. RESULTS: Ten patients with ages ranging from 1 day to 3 years (mean age 10.5 months) were enrolled in this study. The average procedure length was 82.5 min. The mean core body temperature throughout the procedure for all-comers was 36.5 ± 0.9 °C with an overall mean difference in maximum and minimum temperatures of 1.32 ± 1.1 °C. CONCLUSION: A liquid-warming garment applied to the head of pediatric surgical patients is an innovative and relatively low-cost means to regulate and to maintain the ideal core body temperature of patients undergoing surgical procedures.

The impact of perioperative warm-up jackets on surgical site infection: cost without benefit?

BACKGROUND: On July 1st, 2012, the University of Minnesota Medical Centers adopted a policy requiring all personnel to wear cover jackets in perioperative areas. This policy is based on the Association of Perioperative Registered Nurses recommended practice for cover jacket usage. We hypothesized that the cover jacket policy had no effect on the surgical site infection rate. METHODS: We compared surgical site infection data from 1 year before the policy and 1 year after the policy. Twenty six thousand three hundred procedures were included: 13,302 before the policy and 12,998 after the policy. Rates between periods were compared using the z-test for proportions. RESULTS: The SSI rate precover and postcover jacket policy was 2.42% and 2.76% respectively. The P value was .1998. Our hypothesis was rejected because the change in rate was not statistically significant. CONCLUSIONS: This study demonstrates that there was not a decrease in SSI rates with this cover jacket policy; in fact, the data show a trend toward an increase in SSI rate thus making the argument for the abandonment of the cover up jackets.

A phase I clinical study to evaluate safety of orally administered, genetically engineered Salmonella enterica serovar Typhimurium for canine osteosarcoma.

We conducted a prospective phase I study to evaluate safety of an orally administered Salmonella encoding IL-2 (SalpIL2) in combination with amputation and adjuvant doxorubicin for canine appendicular osteosarcoma. Efficacy was assessed as a secondary measure. The first dose of SalpIL2 was administered to 19 dogs on Day 0; amputation was done after 10 days with chemotherapy following 2 weeks later. SalpIL2 was administered concurrent with chemotherapy, for a total of five doses of doxorubicin and six doses of SalpIL2. There were six reportable events prior to chemotherapy, but none appeared due to SalpIL2. Dogs receiving SalpIL2 had significantly longer disease-free interval (DFI) than a comparison group of dogs treated with doxorubicin alone. Dogs treated using lower doses of SalpIL2 also had longer DFI than dogs treated using the highest SalpIL2 dose. The data indicate that SalpIL2 is safe and well tolerated, which supports additional testing to establish the potential for SalpIL2 as a novel form of adjuvant therapy for dogs with osteosarcoma.

Vasculature Disruption Enhances Bacterial Targeting of Autochthonous Tumors.

Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has been developed as a vector to deliver therapeutic agents to tumors. The potential of S. Typhimurium in cancer therapy is largely due to its reported propensity to accumulate at greater than 1,000-fold higher concentrations in tumors relative to healthy tissues. In this study, we compared bacterial colonization of tumors in a subcutaneous transplantation model with a more clinically relevant autochthonous tumor model. Following intravenous administration of attenuated S. Typhimurium strain SL3261, we observed approximately 10,000-fold less bacteria in autochthonous tumors that sporadically develop in transgenic BALB-neuT mice compared to tumors developed from subcutaneous transplantation of 4T1 murine breast cancer cells in BALB/c mice. Treatment of BALB-neuT mice with a vasculature-disrupting agent (VDA) prior to bacterial treatment caused necrosis of tumor tissue and significantly increased the bacterial targeting of autochthonous tumors by approximately 1,000-fold. These observations emphasize the importance of appropriate model selection in developing bacteria-based cancer therapies and demonstrate the potential of combining VDA pre-treatment with bacteria to facilitate targeting of clinically relevant tumors.

Expression of periplasmic chaperones in Salmonella Typhimurium reduces its viability in vivo.

The efficacy of live attenuated bacterial vectors is dependent upon the fine-tuning of a strain's immunogenicity and its virulence. Strains are often engineered to deliver heterologous antigens, but soluble expression of recombinant proteins can be troublesome. Therefore, secretion systems or chaperone proteins are routinely used to assist in attaining high levels of functional, soluble protein production. However, the effects of chaperone expression on the virulence of attenuated bacterial vectors have not been previously reported. In anticipation of utilizing periplasmic chaperone proteins to facilitate soluble production of immunomodulatory proteins in an attenuated strain of Salmonella Typhimurium, the production of the chaperones was tested for their effect on both culture growth and bacterial persistence in mouse tissues. Although no effect on growth of the bacteria was observed in vitro, the increased expression of the periplasmic chaperones resulted in over-attenuation of the Salmonella in vivo.

Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli.

Programmed death ligand 1 (PD-L1), is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. Single chain variable fragments (scFvs) have several desirable characteristics and are an attractive alternative to monoclonal antibodies for experimental or therapeutic purposes. Three chickens were immunized against murine PD-L1, and mRNA isolated from their spleens was used to generate an immunized immunoglobulin variable region library. Using splice-overlap extension PCR, variable region cDNAs were combined to generate full-length scFvs. M13 phage display of the resulting scFv library identified a functional scFv against PD-L1 (αPD-L1 scFv). The scFv was expressed as soluble protein in the periplasm and culture supernatant of recombinant Escherichia coli and purified with a 6×-His tag using immobile metal affinity chromatography. The dissociation constant of αPD-L1 scFv was determined to be 7.11×10(-10)M, and the scFv demonstrated inhibitory biological activity comparable to an antagonistic monoclonal antibody, providing an alternative agent for blocking PD-1/PD-L1 signaling.

Antioxidant oils and Salmonella enterica Typhimurium reduce tumor in an experimental model of hepatic metastasis.

Fruit seeds high in antioxidants have been shown to have anticancer properties and enhance host protection against microbial infection. Recently we showed that a single oral dose of Salmonella enterica serovar Typhimurium expressing a truncated human interleukin-2 gene (SalpIL2) is avirulent, immunogenic, and reduces hepatic metastases through increased natural killer cell populations in mice. To determine whether antioxidant compounds enhance the antitumor effect seen in SalpIL2-treated animals, we assayed black cumin (BC), black raspberry (BR), and milk thistle (MT) seed oils for the ability to reduce experimental hepatic metastases in mice. In animals without tumor, BC and BR oil diets altered the kinetics of the splenic lymphocyte response to SalpIL2. Consistent with previous reports, BR and BC seed oils demonstrated independent antitumor properties and moderate adjuvant potential with SalpIL2. MT oil, however, inhibited the efficacy of SalpIL2 in our model. Based on these data, we conclude that a diet high in antioxidant oils promoted a more robust immune response to SalpIL2, thus enhancing its antitumor efficacy.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System.

BACKGROUND: Metastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver. RESULTS: Liver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 µg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 µg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 µg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid. CONCLUSION: These results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

Retrospective review of reoperative pectus excavatum repairs.

BACKGROUND/PURPOSE: Despite success of several techniques described for pectus excavatum repair, a minority of patients require multiple reoperations for recurrence or other complications. We aimed to review our experience in reoperative pectus excavatum repairs and to identify features correlating with need for additional reoperations. METHODS: Charts were reviewed of all patients undergoing reoperative pectus excavatum repair for 3 years at a university-based children's hospital. Number and type of previous repairs, time between operations, lengths of stay, analgesia, and complications were recorded. RESULTS: From February 2004 to December 2007, 170 pectus excavatum repairs were performed. Among these, 27 were reoperative. Overall, 18.2% of reoperative patients required subsequent additional reoperations. 21.1% of patients undergoing repeat open repairs and 33.3% of patients undergoing repeat minimally invasive repairs required further operative interventions. There was no need for additional repairs among patients who had open repairs after minimally invasive repairs, nor for any patients who had minimally invasive repairs after open repairs. CONCLUSIONS: We conclude that patients with failed open repairs will have better success with minimally invasive reoperations, whereas patients with failed minimally invasive repairs will have better success with open reoperations. When faced with reoperative pectus excavatum, we recommend consideration of an alternative operative approach from the initial procedure.

Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo.

BACKGROUND: Heat shock protein (Hsp)-70 is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. Our objectives were to assess the effectiveness of triptolide, an Hsp-70 inhibitor, in treating neuroblastoma in vitro and in vivo, and to measure the associated effects on Hsp-70 levels and apoptosis markers. METHODS: After exposing N2a and SKNSH cell lines to triptolide, cell viability was assessed. Caspase-3 and -9 activities were measured and annexin staining performed to determine if cell death occurred via apoptosis. Hsp-70 protein and mRNA levels were determined using Western blot and real-time polymerase chain reaction. In an orthotopic tumor model, mice received daily triptolide injections and were humanely killed at study completion with tumor measurement. RESULTS: Triptolide treatment resulted in dose- and time-dependent N2a cell death and dose-dependent SKNSH killing. Triptolide exposure was associated with dose-dependent increases in caspase activity and annexin staining. Triptolide decreased Hsp-70 protein and mRNA levels in a dose-dependent fashion. Mice receiving triptolide therapy had significantly smaller tumors than controls. CONCLUSION: Triptolide therapy decreased neuroblastoma cell viability in vitro and inhibited tumor growth in vivo. Our studies suggest that triptolide killed cells via apoptosis and in association with inhibition of Hsp-70 expression. Triptolide may provide a novel therapy for neuroblastoma.