Shawn P. Fagan

Insulinoma-induced hypoglycemic death in mice is prevented with beta cell-specific gene therapy.

Objective and Summary Background DataTumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/ SCID mouse model to prevent hypoglycemic death. MethodsRat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase–polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored. ResultsOnly NIT-1 cells stained blue after X-gal staining or had detectable levels of &bgr;-galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia. ConclusionsThe data suggest that the RIP is an insulinoma-specific promoter. An ICR/ SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.

Spider Bites Presenting with Methicillin-Resistant Staphylococcus aureus Soft Tissue Infection Require Early Aggressive Treatment

BACKGROUND Occasionally, spider bites result in necrotizing soft tissue infections that require aggressive surgical debridement and treatment with intravenous antibiotics. With the rise of microbial resistance in the community, management with standard gram-positive intravenous antibiotic coverage may be ineffective. Our objective was to determine the infectious organisms cultured following wide local excision of soft tissue infections caused by spider bites. We hypothesized that the majority of isolated organisms would be sensitive to penicillin based antibiotics. METHODS From March 2000 to November 2001, the medical records were reviewed of patients who presented to a tertiary care hospital with serious soft tissue infections secondary to spider bites that required surgical treatment. For each patient, demographics, symptoms, size, time to surgical evaluation (TTSE), temperature, white blood cell (WBC) count, surgical procedure, and culture data were collected. Data are presented as mean +/- SEM. RESULTS Thirty-eight patients presented with serious soft tissue infections secondary to spider bites that required surgical debridement and treatment with intravenous antibiotics. Twenty-nine percent (11 of 38) of these patients had failed initial outpatient therapy with penicillin-based oral antibiotics. The mean TTSE was 5.0 +/- 0.5 days (range = 2-14 days; median = 4.5 days). The most common presenting symptoms were pain and erythema surrounding the bite site. The mean temperature was 98.8 +/- 0.6 degrees F (range = 97.2-102.2 degrees F; median = 99.2 degrees F). The mean WBC count was 12.6 +/- 0.8 mm3. All patients required wide surgical debridement of the infected area. The mean size of the excised tissue was 26 +/- 4 cm2 (range = 4-120 cm2; median = 16 cm2). Every patient had cultures that grew Staphylococcus aureus. In 86.8% of patients, S. aureus was found to be methicillin-resistant (MRSA). All isolated organisms were sensitive to trimethoprim-sulfamethoxazole. CONCLUSIONS In our experience, patients who presented with soft tissue infections as result of spider bites predominantly had methicillin-resistant S. aureus infections, corresponding to the increased incidence of MRSA reported in the community. Therefore, a more aggressive approach to the management of spider bites presenting with severe cellulitis is warranted. Routine treatment should include aggressive surgical debridement, intraoperative wound cultures, the empiric use of antibiotics with activity against MRSA, and adjustment of antimicrobial therapy based on culture and sensitivity data.

The Cre-loxP system: a versatile tool for targeting genes in a cell- and stage-specific manner.

Gene-targeted mice, derived from embryonic stem cells, are useful tools to study gene function during development. However, if the inactivation of the target gene results in embryonic lethality, the postdevelopmental function of the gene cannot be further studied. The Cre recombinase-loxP (Cre–loxP) system was developed to overcome this limitation as well as to confine the inactivation of the target gene in a cell- or tissue-specific manner. This system allows for the inactivation of the target gene in a single cell type, thereby allowing the analysis of physiological and pathophysiological consequences of the genetic alteration in mature animals. A unique property of the insulin gene to be expressed only in pancreatic beta cells has allowed using the beta-cell-specific rat insulin promoter (RIP) for Cre recombinase expression to inactivate genes in beta cells. The RIP has been used to inactivate genes in beta cells and analysis of these genetically altered mice has provided important information regarding the role of potential transcription factors and the receptors in vivo, for regulation of insulin gene transcription and in the development of beta cells. The Cre–loxP system is at a relatively early stage of development, and the ability of this technique to virtually target any gene in any tissue at any stage of development makes the study of gene function in a single cell type in vivo an attainable goal. It is anticipated that the continued experience with this system will provide an important tool to determine the role of the transcription factors involved in insulin gene regulation and islet cell differentiation and ultimately provide the basis for novel therapy to treat diabetes.

Activation of somatostatin receptor subtype 2 inhibits insulin secretion in the isolated perfused human pancreas.

Objectives Five distinct somatostatin receptors (SSTRs) have been cloned, characterized, and designated SSTRs 1–5. The role of these receptors in B-cell signaling has not been well characterized. Methods In the current study, the isolated perfused human pancreas model was used to determine the specific effect of 4 different somatostatin receptor agonists on insulin secretion. Conclusion We demonstrated that the SSTR 2 agonist and octreotide significantly suppressed insulin secretion. Furthermore, even during the immunoneutralization of endogenous intrapancreatic somatostatin, the SSTR 2 agonist was able to reverse the effect of somatostatin immunoneutralization by suppressing insulin secretion. These results demonstrate that activation of SSTR 2 suppresses insulin secretion in the isolated perfused human pancreas.

Acute respiratory failure after pleurodesis with doxycycline

Bedside pleurodesis through a tube thoracostomy has been shown to be effective treatment of malignant pleural effusion and pneumothorax with persistent air leak. A variety of agents can be used, and each has been shown to produce rare but potentially serious complications. We report a case of sudden, severe respiratory failure in a 42-year-old man after pleurodesis with 300 mg of doxycycline. His response was consistent with an anaphylactic reaction. After intubation, mechanical ventilation and nebulizer treatments, he rapidly recovered to baseline. On the basis of this report and a review of the literature, we believe that doxycycline may not be an innocuous agent for bedside pleurodesis and that such procedures warrant a monitored setting.

Development of a transgenic mouse model using rat insulin promoter to drive the expression of CRE recombinase in a tissue-specific manner

SummaryBackgroundTissue-specific ablation of a gene using the Cre-loxP system has been used as an important tool to define its role, in addition to the total ablation, to avoid the embryonic lethality in case of wide expression of the target gene.MethodsThe RIP-Cre genetic construct was generated by standard subcloning techniques and microinjected into one cell embryo to develop the transgenic mouse line. Transgenic mice were screened by polymerase chain reaction (PCR) using DNA isolated from tell digestion. Tissue specificity of RIP was demonstrated by transient transfection of RIP-lacZ construct to NIT-1 cells (mouse insulinoma cell line) in vitro.ResultsThe 448 nucleotides of RIP were sufficient for β-cell specific expression of the reporter gene as evidenced by the presence of blue color in the nucleus of NIT-1 cells. Isolated RIP-Cre transgene was microinjected, and PCR screening identified two independent lines of transgenic mice. Tissue specificity of RIP was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) using the islet RNA from the transgenic mice.ConclusionWe have established a tissue-specific transgenic mouse model using Cre recombinase linked to rat insulin promoter (RIP) to drive the expression of the reporter gene specifically in the β-cells. The RIP-Cre transgenic mice will allow β-cell specific ablation of target gene(s) to define its role in the regulation of islet physiology.