Gene transfer to schwann cells after peripheral nerve injury: A delivery system for therapeutic agents

We transferred a reporter gene to Schwann cells to test whether they might serve as an endoneurial delivery system for therapeutic proteins. A replication-defective adenoviral vector carrying the gene for β-galactosidase (lacZ) was injected into the distal segment of intact or crushed sciatic nerves of adult rats, and the expression of lacZ was histochemically assessed. Less than 1% of the Schwann cells became reactive in intact nerves, but up to 18% of the proliferating Schwann cells of injured nerves expressed lacZ. Gene expression decayed with time but might persist for up to 2 months. It was enhanced by immunosuppression: daily cyclosporin A injections reduced both proliferation of Schwann cells and lymphocytic infiltration of the nerve, whereas tolerance induced by a single intrathymic injection of the vector 4 days after birth abolished the inflammatory response but not the proliferation of Schwann cells. The vector itself did not impede axonal regeneration. The results indicate that adenoviral gene transfer to Schwann cells in injured nerves is possible and suggest that induced production of neurotrophic factor may represent a therapeutic supplement to surgical nerve repair.     

Scheduling of combination chemotherapy for a murine melanoma,with the subrenal capsular assay

The effect of varying schedules for administration of two drugs, dimethyl triazeno imidazole carboxamide (DTIC) and methylcyclohexyl chloroethyl nitrosourea (MeCCNU), was studied using the Harding-Passey Mouse melanoma in the subrenal capsular assay. The results of the assay indicate that the responses of the tumor varied depending on the schedule of administration. The best response, with a mean decrease in dimensions of 3.0 ocular micrometer units (± 1.12 SD), was noted with administration of DTIC on day 1 after tumor implantation, followed by MeCCNU on day 2. These results are similar to previously reported data in CD2F1 mice bearing intraperitonal Harding-Passey melanoma and suggest that the subrenal capsular assay may be of use in determining the best combinations and schedules of administration for treatment of tumors.     

Concentric joint space narrowing of the hip associated with hemosiderotic synovitis (HS) including pigmented villonodular synovitis (PVNS)

Concentric joint space narrowing of the hip is an expected radiographic finding in cases of inflammatory arthritis such as rheumatoid arthritis or sepsis. However, similar joint space narrowing is associated with chronic hemorrhagic conditions that produce hemosiderotic synovitis. Hemosiderotic synovitis results from chronic intraarticular bleeding such as occurs in pigmented villonodular synovitis, generalized bleeding diathesis, synovial hemangioma, and chronic trauma. Five hips in five patients with concentric joint space narrowing not associated with inflammatory arthritis or with hemophilia were reviewed clinically, radiographically, and pathologically. All patients had a hemosiderotic synovitis. The definitive diagnosis of pigmented villonodular synovitis was made pathologically in two cases that demonstrated nodular areas of giant cell proliferation, collagen production, and lipid-laden histiocytes on histologic samples.     

Comparison of the rate of aminoacylation of tRNA isolated from NMRI mouse liver with tRNA isolated from Krebs II ascites or mouse plasmacytoma cells

A comparison of the initial rates of aminoacylation of tRNAs isolated from different sources for 17 amino acids was peformed. tRNA was isolated from NMRI mouse liver (tRNA L) and from Krebs II ascites tumors (tRNA Asc), and aminoacyl-tRNA synthetases were prepared from the latter cells. The aminoacylation of tRNA Asc was 31–88% slower than the charging of tRNA L.In similar studies, tRNA from a mouse plasmacytoma tumor (tRNA Mt) and from suspension cultured cells of the same cell line (tRNA M) were compared to tRNA L in the aminoacylation reaction catalysed by synthetases isolated from tumor or suspension cultured cells. About half of the tRNAs (Mt or M) for the 17 amino acids tested differed in charging rate when compared to tRNA L, but the differences were not as great as those observed in the experiments where tRNA Asc and tRNA L were compared.     

Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n = 11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.     

In vitro activation of cyclophosphamide for an in vitro chemosensitivity assay

The problem of activation of cyclophosphamide (CY) for an in vitro chemosensitivity assay was studied using the B16 melanoma. The efficacy of the S9 hepatic microsomal fraction in vitro was compared with activation by passage of drug in vivo. The effect of CY was assayed by inhibition of tritiated thymidine (³HdThd) uptake by B16 tumor cells in vitro, and by its effect in single dose in vivo on the life span of syngeneic C57BL/6 mice injected with B16 tumor cells. In vivo activation of CY that was achieved by injecting 20 mg of CY intraperitoneally (i.p.) into mice and obtaining plasma 20 minutes later was more rapid and more reproducible, while in vitro activation provided a more potent preparation and a better quantitative correlation with the in vivo effect of CY. The activation activity of different preparations of S9 microsomal fractions was found to be directly related to the activity of aminopyrine demethylase, a mixed function oxidase enzyme, in the S9 fraction, rather than to total protein concentration. The S9-activated drug required 2 1/2 to 3 hours to achieve maximum inhibition of subsequent tumor cell tritiated thymidine (³HdThd) incorporation compared with 20 minutes to achieve maximum inhibition by in vivo activated drug. We conclude that rapid qualitative screening for effectiveness of CY may best be done with in vivo activated drug, whereas quantitative prediction of effective concentration appears to be best achieved with drug activated in vitro by the hepatic S9 fraction.     

A Randomized, Controlled Trial Comparing Long-term Cosmetic Outcomes of Traumatic Pediatric Lacerations Repaired with Absorbable Plain Gut versus Nonabsorbable Nylon Sutures

OBJECTIVES: To show that the use of absorbable sutures in pediatric traumatic lacerations affords good long-term cosmesis and no increase in complications (infection, dehiscence rates, and need for surgical scar revision) when compared with wounds sutured with nonabsorbable sutures. METHODS: This was a randomized clinical trial conducted in a pediatric emergency department. Patients 1-18 years of age who presented to the emergency department with lacerations < 12 hours old were recruited between January 1999 and December 2001. Exclusion criteria were the following: wounds that could be approximated by tissue adhesives, animal/human bites, gross contamination, puncture/crush wounds, wounds crossing joints, lacerations of tendon/nerve/cartilage, collagen vascular disease, immunodeficiency, diabetes mellitus, bleeding disorder, and scalp lacerations. Patients were randomized into one of two groups: those receiving absorbable plain gut sutures (group A) and those receiving nonabsorbable nylon sutures (group NA). Board-eligible/certified pediatric emergency physicians or clinical fellows performed laceration repair in a standardized approach. All wounds were reevaluated within ten days by a single research nurse who assessed the wounds using a previously validated wound evaluation score (WES) composed of six items (presence of step-off, contour irregularities, margin separation, edge inversion, extensive distortion, and overall cosmetic appearance). A score of 6/6 was considered optimal. The study nurse also noted the presence of infection and dehiscence. The patients were then seen by a single blinded plastic surgeon at four or five months who evaluated the wound using a previously validated visual analog scale of cosmesis (VAS). In addition, the surgeon repeated the WES and assessed the need for surgical scar revision. RESULTS: A total of 147 patients were eligible, and 52 patients declined to participate. Of the 95 patients enrolled, 50 were randomized to group A and 45 to group NA. The two groups had similar ages, gender distributions, rates of use of sedation or steri-strips, wound lengths and widths, mechanisms of injury, and wound locations. At the short-term follow-up, no difference was found in the proportion of optimal WES scores between group A (63% of patients) and group NA (49% of patients) (relative risk = 0.73; 95% confidence interval [95% CI] = 0.45 to 1.17). No difference was found in the rates of infection and dehiscence between the two groups. Sixty-three of the 95 patients presented for long-term follow-up. The groups remained similar with respect to patient and wound characteristics as well as wound location. The average VAS score at four months was 79 (95% CI = 73 to 85) for group A and 66 (95% CI = 55 to 76) for group NA. In addition, no differences were found in the proportion of optimal WES between group A (36% of patients) and group NA (28% of patients) at four months (relative risk = 0.88; 95% CI = 0.62 to 1.26). Surgical scar revision was recommended for only three patients, of whom two were in group A. No patients chose to have their scars revised. No differences were found between group A and group NA for the rates of dehiscence (2% vs. 11%; p = 0.07) and infection (0 vs. 2; p = 0.3). CONCLUSIONS: The use of plain catgut absorbable sutures in the repair of traumatic lacerations in children appears to be an acceptable alternative to nonabsorbable sutures because the long-term cosmetic outcome seems to be at least as good. In this study, plain gut suture material seemed to provide slightly better cosmesis. In addition, no difference was found in the rate of dehiscence or infection between the groups.     

Multimodal hybrid imaging agents for sentinel node mapping as a means to (re)connect nuclear medicine to advances made in robot-assisted surgery

Purpose

Radical prostatectomy and complementary extended pelvic lymph node dissection (ePLND) of sentinel lymph nodes (SNs) and non-sentinel lymph nodes (LNs) at risk of containing metastases are increasingly being performed using high-tech robot-assisted approaches. Although this technological evolution has clear advantages, the physical nature of robotic systems limits the integrated use of routine radioguided surgery technologies. Hence, engineering effort in robotics are focused on the integration of fluorescence guidance technologies. Using the hybrid SN tracer indocyanine green-^99mTc-nanocolloid (radioactive and fluorescent), for the first time in combination with a robot-integrated laparoscope, we investigated whether the robot-assisted approach affects the accuracy of fluorescence detection of SNs identified preoperatively using nuclear medicine.

Methods

The study included 55 patients (Briganti nomogram-based risk >5 % on LN metastases) scheduled for robot-assisted radical prostatectomy, SN biopsy and ePLND. Following indocyanine green-^99mTc-nanocolloid injection, preoperative nuclear imaging (lymphoscintigraphy and SPECT/CT) was used to locate the SN(s). The fluorescence laparoscope was used intraoperatively to identify the SN(s) with standard fluorescence settings (in 50 patients) and with customized settings (in 5 patients). The number and location of the SNs, the radioactive, fluorescence (both in vivo and ex vivo) and tumour status of the resected SNs/LNs, and postoperative complications were recorded and analysed.

Results

Combined, preoperative lymphoscintigraphy and SPECT/CT imaging identified 212 SNs (median 4 per patient). Intraoperative fluorescence imaging using standard fluorescence settings visualized 80.4 % (148/184 SNs; 50 patients; ex vivo 97.8 %). This increased to 85.7 % (12/14 SNs; 5 patients; ex vivo 100 %) with customized fluorescence settings. SPECT/CT images provided guidance towards the residual SNs. Ex vivo all removed SNs were radioactive. SNs were tumour-positive in 25.4 % of patients (14/55; false-negative rate 7 %, 1/14 patients). In ten patients, the SN was the only tumour-positive LN. Surgical complications were minimal.

Conclusion

Directly linking 3D preoperative nuclear imaging information on SNs to a robot-integrated fluorescence laparoscope improved the surgeon’s use of the technology and did not influence the sensitivity or morbidity of the procedure. To our surprise, however, the detection rates with the current fluorescence camera did not improve.