High efficacy of integrated preventive measures against zoonotic cutaneous leishmaniasis in northern Afghanistan, as revealed by Quantified Infection Rates

Following an outbreak of more than 200 cases of zoonotic cutaneous leishmaniasis (ZCL) during 2004 and 2005 among International Security Assistance Force (ISAF) troops stationed in the Mazar-e Sharif (MeS) airport area, epidemiological investigations indicated the presence of a local high-density ZCL focus. Based on ZCL's specific transmission modes, density and seasonality, integrated preventive measures were continuously implemented from February 2006 at a German military camp constructed at MeS. Preventive techniques included such synergistic measures as skin repellents and insecticide-impregnated clothing, bednets and curtains, sand fly and rodent monitoring and control, extended habitat sanitation, and health education, all designed to achieve maximum protection against ZCL. Habitat alteration included: (a) erection of a 3.0 m high stone wall around the entire camp area; (b) removal of ≥30 cm of the upper earth layer throughout the site; (c) soil compaction and stone paving to a depth of ≥30 cm plus compaction of the surrounding area to a distance of 100 m outside the camp wall; and (d) regular eradication of vegetation. Aggressive implementation of these measures led to a 166-fold and 546-fold reduction in sand fly numbers and complete eradication of the local rodent reservoir, Rhombomys opimus, inside the camp during 2006 and 2007, respectively. ZCL attack rates decreased significantly (p < 0.0001), from 17.5% (14 cases/80 persons) for the 2005 German assessment teams to 0.087% (1 case/1150 persons) for the 2006 contingent, and 0% (0 cases) (p < 0.0001) for the 2007 contingent, with Quantified Infection Rates (QIRs) of 0.058, 0.0000055, and 0.0, respectively. Using QIR values, the protective factor of the integrated preventive measures was shown to be ≥10,545 times higher in the 2006 and 2007 contingents, compared with the 2005 assessment teams. Results show that the continuously implemented, integrated preventive techniques used in this study gave excellent and long-lasting protection against zoonotic cutaneous leishmaniasis under field conditions.     

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery.

Background: Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies. Aim: For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals. Results: We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22kDa linear PEI (L-PEI), the low-molecular weight (4-10kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection. Conclusions: Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.     

Glucuronidation of carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Since carcinogenic arylamines are sequentially oxidized andconjugated with glucuronic acid, differences in glucuronidationmay critically determine the toxic potential of these compounds.Therefore, N-glucuronldation of 1- and 2-naphthylamine (1-NAand 2-NA), 4-aminobiphenyl (4-ABP) and their N-hydroxy derivativeswas investigated using rat and human liver microsomes and V79cell-expressed phenol UDP-glucuronosyltransferases (UGT) ofthe UGTl gene complex. Cell-expressed UGTs included rat andhuman UGT 1.6, which are known to conjugate planar phenols,and human UGT1.7, conjugating both planar and bulky phenols,(i) N-Glucuronidation of 1- and 2-NA and of N-hydroxy-2-NA wasinducible by 3-methylcholanthrene in rat liver microsomes whereasN-glucuronidation of the bulky arylamines 4-ABP and N-hydroxy-4-ABPwas not In support of these findings mutagenicity of N-hydroxy-2-NAin the Ames test was markedly reduced upon addition of UDP-glucuronicacid using liver homogenates from 3-methylcholanthrene-treatedrats, (ii) With cell-expressed rat UGT1.6, non-carcinogenic1-NA was conjugated with the highest rate and with higher affinitythan 2-NA. UGT1.6 showed poor activity towards N-hydroxy-4-ABPand 4-ABP. (iii) Substrate specificity of human UGT1.6 alsoappeared to be limited to planar 1-NA, 2-NA and its N-hydroxyderivative, whereas UGT1.7 showed broader substrate specificity,including the bulky arylamine 4-ABP and its /V-hydroxy derivative.The results suggest marked differences in substrate specificityof different UGT isozymes for arylamines and their N-hydroxyderivatives.     

Endomyocardial biopsy in infants and children: Experience in 60 patients

Summary In 60 children, aged between 1 month and 22 years (median 3.54 years) and with a body weight of 3–67 kg (median 12.6 kg), transvascular endomyocardial biopsy (EMB) was performed from the right (35 children) or left ventricle (30 children). The specimens were investigated by light and electron microscopy. There were three indications for biopsy: (1) poorly functioning, dilated left ventricle (seven patients with endocardial fibroelastosis, 16 with dilated cardiomyopathy, six from healing/healed or chronic myocarditis); (2) unexplained left ventricular hypertrophy (10 with hypertrophic cardiomyopathy, four with secondary hypertrophy, three with storage diseases); (3) to answer certain questions in eight children (four with hypoxic and two with cytoxic myocardial damage). Retrospectively, there were five nonindicated biopsies. There were no serious complications. Biopsies were diagnostic in 11.7% of cases, helpful in 71.7%, and of no help in 16.6%. Thus even in childhood endomyocardial biopsy is a diagnostic tool which can add useful information on the etiology or pathogenesis of an underlying myocardial disease.     

Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU

O⁶-Alkylguanine-DNA alkyltransferase (O⁶-AGT) activity in rat ovarian tumor lines O-342 and O-342/DDP was 103.4±18.4 and 240.9±40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O⁶-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O⁶-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O⁶-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O⁶-AGT activity was 337.6±18.2 and 180.0±39.9 fmol/mg protein, respectively; in these lines depletion of O⁶-AGT activity by O⁶-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O⁶-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.Abbreviations BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) - CENU 2-chloroethylnitrosourea - DDP cisplatin ED₅₀, the effective dose required to inhibit colony formation or cell proliferation by 50% - O⁶-AGT O⁶-alkylguanine-DNA alkyltransferase - O⁶-BG O⁶-benzylguanine SF sensitization factor     

Accuracy of frozen section examination of testicular tumors of uncertain origin

INTRODUCTION: A total of 80-90% of all testicular masses are malignant germ cell tumors. Benign testicular lesions are recognized in approximately 10-20% enabling a testis-preserving surgery on the findings of frozen section examination (FSE). However, there are only sparse information with regard to the reliability of FSE in testicular tumors of uncertain dignity. Therefore, we retrospectively reviewed our experience concerning the reliability of FSE in primary testicular tumors by comparing each FSE result to the final diagnosis. PATIENTS AND METHODS: From 1974 to 2000, 354 patients were operated on a testicular tumor. During inguinal exploration and after clamping of the spermatic cord and appropriate dressing, a representative biopsy of the tumor was taken and sent for FSE. In case of malignancy radical orchiectomy was performed, in case of benign findings or in case of a germ cell tumor in a solitary testicle, the tumor was enucleated. Slides of FSE and the permanent sections were reviewed and compared with regard to the histological diagnosis and presence/absence of malignancy. RESULTS: Based on FSE, 317 tumors (89.5%) were found to be malignant ((100 seminomas (38.5%), 217 nonseminomas (61.5%)) and 37 tumors (10.5%) were benign (17 epidermoid cysts, 14 Leydig cell tumors, two cystadenomas, two simple cysts, two hemangiomas). Comparing FSE and definitive diagnosis, FSE correctly identified all malignant and benign lesions. There was a failure rate of 10 and 8% to differentiate seminomatous from nonseminomatous tumors and vice versa based on FSE, which, however, was irrelevant for the surgical management. Complications of the enucleations (n = 37) were: testicular atrophy in three cases, testicular hematoma in three cases, orchitis/epididymitis in one case. Not a single case disclosed a local relapse after a mean follow-up of 105 (12-240) months. CONCLUSIONS: Intraoperative FSE correctly identified all malignant and benign testicular masses including radical orchiectomy or organ-preserving surgery. Surgical management of testicular tumors based on FSE results is clinically practicable.     

Three-axis ellipsoidal fitting of videokeratoscopic height data after penetrating keratoplasty

PURPOSE: After penetrating keratoplasty corneal topography tends to be irregular and the fitting of spectacle glasses or contact lenses may be difficult. The purpose of this study was to demonstrate a mathematical method for approximation of discrete corneal topography height data with an ellipsoid for better appreciation of the clinical outcome after PK. PATIENTS AND METHODS: In 50 eyes (30 keratoconus, 20 Fuchs' dystrophy) penetrating keratoplasty was performed using nonmechanical trephination with the excimer laser 193 nm. Main outcome measures were objective corneal astigmatism (regular keratometry, corneal topography (TMS-1)), subjective refraction and best-corrected visual acuity (VA) in a fixed postoperative gate 3 and 12 month postoperatively and after suture removal. An approximation algorithm was applied for fitting a general ellipsoidal surface (not rotationally symmetric) to raw corneal topography height data. A set of parameters (meridional power, axis and asphericity) were calculated. The root mean square error (RMS) was determined between raw topography power data and the ellipsoidal model surface within an apical distance of 3 mm. The cylinder of subjective refraction was correlated with the keratometric readings, the Simulated Keratometry (SimK) of the topography system and the respective parameters of the model surface. RESULTS: The amount of the SimK cylinder yielded higher values than keratometry and the ellipsoidal fit; subjective refraction yielded the lowest value at each follow-up interval. The ellipsoidal fit showed the best correlation to the refractive cylinder at all follow-up stages (p = 0.04 at 3, p = 0.01 at 12 months and p = 0.002 after suture removal). The axis of the best ellipsoidal fit showed a significant correlation with the axis of the refractive cylinder at all follow-up intervals (p = 0.02 at 3 months, p = 0.01 before suture removal and p = 0.002 after suture removal). The axis of the keratometric cylinder showed a mild correlation at all follow-up examinations (p = 0.05 at 3 months, p = 0.02 before suture removal and p = 0.04 after suture). The cylinder of the topographic modeling system, however, showed a significant correlation with the refractive cylinder axis only after suture removal (p = 0.04). The paracentral corneal power of SimK (45.9D at 3 months, 44.4D at 12 months and 43.0D after suture removal) exceeded the respective values of conventional keratometry (43.1D at 3 months, 42.9D at 12 months and 41.7D after suture removal) and the ellipsoidal fit (43.3D at 3 months, 43.0D at 12 months and 41.8D after suture removal). The corneal asphericity from the ellipsoidal fit reached an approximately spherical shape in radial direction (A = 1.0) in the initial time period after penetrating keratoplasty, remained stable before suture removal and decreased significantly (p = 0.02) to a final value of A = 0.86 indicating a (normal) prolate shape of the cornea. The approximation error between the raw corneal topography height data and the best ellipsoidal fit model surface was nearly unchanged before suture removal (1.8 +/- 0.7 microm at 3 months and 1.9 +/- 1.1 microm at 12 months, p = 0.30) and decreased significantly to the examination after suture removal (0.9 +/- 0.5 microm, p = 0.01). CONCLUSIONS: The approximation of corneal topography height data with an ellipsoidal model surface renders reconstruction of clinically relevant corneal topography parameters including corneal asphericity. Even in markedly irregular corneal surfaces, such as after PK, the correlation of amount/axis of refractive cylinder with the model surface parameters is more accurate than with respective SimK values of corneal topography analysis.     

Superficial corneal effects of experimental nonmechanical penetrating keratoplasty using a Q-switched Er:YAG laser

PURPOSE: To assess thermal effects of Q-switched Er:YAG laser trephination to corneal epithelium and superficial stroma using different mask types and materials for experimental penetrating keratoplasty. METHODS: Laser trephination was performed in 20 freshly-enucleated porcine eyes (repetition rate 5 Hz, pulse energy 65 mJ, spot size 0.7 mm). We used flat, open-metal and ceramic masks for donor and recipient trephination placed directly onto the corneal surface. Main outcome measures as assessed by light microscopy after PAS staining of 8-microm paraffin sections included: extension of tissue thermal damage at the cut edge in the superficial and basal epithelial layers, the basement membrane and subepithelial stroma, and depth and width of epithelial/stromal involvement in the area of the donor mask contact. RESULTS: The thermal damage in the superficial epithelium was more pronounced in donor (mean extension 61.6 +/- 15.6 microm) than in recipient (29.4 +/- 24.9 microm, p= 0.05) trephination. In donor trephination, thermal damage zone of the superficial epithelial layer was significantly smaller with ceramic than with metal masks (21.0 +/- 23.0 versus 61.6 +/- 15.6 microm, p= 0.014). In contrast, differences at basal epithelial layer (p= 0.44), basement membrane (p= 0.79), and subepithelial stroma (p= 0.2) were not statistically significant. Superficial donor involvement of the cornea adjacent to the paracentral donor mask contact zone was seen neither with ceramic nor with metal masks. CONCLUSION: Superficial corneal alterations adjacent to the mask-cornea contact zone may be minimized by using the Er:YAG laser in a Q-switched mode. Ceramic masks, in contrast to metal masks, further reduce superficial thermal alterations at the cut edge.     

Risk factors for corneal allograft rejection: intermediate results of a prospective normal-risk keratoplasty study

PURPOSE: To analyze the incidence of and possible risk factors for endothelial corneal allograft rejection in a well-defined population following penetrating normal-risk keratoplasty. METHODS: Since 1996 a longitudinal prospective study has been conducted to analyze the results of normal-risk penetrating keratoplasty. All patients underwent a standardized protocol of follow-up treatment and examinations in our institution. Diagnosis of corneal endothelial rejection was based on slit-lamp biomicroscopy and laser flare photometry. Data were analyzed using a proportional hazard model for censored data (Cox model), and Kaplan-Meier survival curves. The following parameters were analyzed: age, gender, atopic dermatitis, dry eye symptoms of the recipient; surgeon, graft diameter, post-mortem time, storage time and graft preservation method; and duration of postoperative epithelial defects. RESULTS: Between 1996 and May 2001, 397 patients were recruited and followed with a median follow-up of 18 months. Episodes of endothelial graft rejection were observed in 22 patients (5.5%; 18 eyes with acute diffuse episodes and 4 eyes with chronic focal rejection episodes). In addition, 12 eyes (3%) showed isolated small keratic precipitates ("graft rejection suspects"). All but one graft regained clarity after topical and systemic steroid treatment. Most episodes occurred 11-18 months postoperatively. The percentage of grafts without any episode of endothelial allograft rejection was 95% after 12 months, 89% after 18 months, and 86.5% after 24 months. The following factors were associated with graft rejection: atopic dermatitis (P=0.021), clinically manifest tear insufficiency (P=0.007), and short duration of graft storage (P=0.008). No significant correlation was detected for the remainder of the analyzed factors (P>0.05). CONCLUSION: The incidence of episodes of corneal endothelial allograft rejection following normal-risk keratoplasty was 13.5% within the first two postoperative years. However, the frequency of irreversible immunologic graft failure (3 per thousand) was lower than reported in the literature. Patients should be regularly followed up for at least 18 months postoperatively. Patients with underlying atopic dermatitis or dry eyes should receive special ophthalmological care.