Expansion of immature, nucleated red blood cells by transient low-dose methotrexate immune tolerance induction in mice.

Biological treatments such as enzyme-replacement therapies (ERT) can generate anti-drug antibodies (ADA), which may reduce drug efficacy and impact patient safety and consequently led to research to mitigate ADA responses. Transient low-dose methotrexate (TLD-MTX) as a prophylactic ITI regimen, when administered concurrently with ERT, induces long-lived reduction of ADA to recombinant human alglucosidase alfa (rhGAA) in mice. In current clinical practice, a prophylactic ITI protocol that includes TLD-MTX, rituximab and intravenous immunoglobulin (optional), successfully induced lasting control of ADA to rhGAA in high-risk, cross-reactive immunological material (CRIM)-negative infantile-onset Pompe disease (IOPD) patients. More recently, evaluation of TLD-MTX demonstrated benefit in CRIM-positive IOPD patients. To more clearly understand the mechanism for the effectiveness of TLD-MTX, non-targeted transcriptional and proteomic screens were conducted and revealed up-regulation of erythropoiesis signatures. Confirmatory studies showed transiently larger spleens by weight, increased spleen cellularity and that following an initial reduction of mature red blood cells (RBCs) in the bone marrow and blood, a significant expansion of Ter-119+ CD71+ immature RBCs was observed in spleen and blood of mice. Histology sections revealed increased nucleated cells, including hematopoietic precursors, in the splenic red pulp of these mice. This study demonstrated that TLD-MTX induced a transient reduction of mature RBCs in the blood and immature RBCs in the bone marrow followed by significant enrichment of immature, nucleated RBCs in the spleen and blood during the time of immune tolerance induction, which suggested modulation of erythropoiesis may be associated with the induction of immune tolerance to rhGAA.

A comparison of cup-to-disc ratio estimates by fundus biomicroscopy and stereoscopic optic disc photography in the Tema Eye Survey.

PurposeTo determine if there are systematic differences in cup-to-disc ratio (CDR) grading using fundus biomicroscopy compared to stereoscopic disc photograph reading.MethodsThe vertical cup-to-disc ratio (VCDR) and horizontal cup-to-disc ratio (HCDR) of 2200 eyes (testing set) were graded by glaucoma subspecialists through fundus biomicroscopy and by a reading center using stereoscopic disc photos. For validation, the glaucoma experts also estimated VCDR and HCDR using stereoscopic disc photos in a subset of 505 eyes that they had assessed biomicroscopically. Agreement between grading methods was assessed with Bland-Altman plots.ResultsIn both sets, photo reading tended to yield small CDRs marginally larger, but read large CDRs marginally smaller than fundus biomicroscopy. The mean differences in VCDR and HCDR were 0.006±0.18 and 0.05±0.18 (testing set), and -0.053±0.23 and -0.028±0.21 (validation set), respectively. The limits of agreement were ~0.4, which is twice as large as the cutoff of clinically significant CDR difference between methods. CDR estimates differed by 0.2 or more in 33.8-48.7% between methods.ConclusionsThe differences in CDR estimates between fundus biomicroscopy and stereoscopic optic disc photo reading showed a wide variation, and reached clinically significance threshold in a large proportion of patients, suggesting a poor agreement. Thus, glaucoma should be monitored by comparing baseline and subsequent CDR estimates using the same method rather than comparing photographs to fundus biomicroscopy.

Coverage of the vitamin A supplementation programme for child survival in Nepal: success and challenges.

BACKGROUND: Nepal's national vitamin A programme, which began in 1993 and continues twice yearly, targets pre-school-aged children in all districts of the country in an effort to reduce morbidity, mortality and nutritional blindness. OBJECTIVE: To characterize the coverage of the Nepal National Vitamin A Programme (NVAP) for pre-school-aged children in Nepal and to identify risk factors for failure to receive vitamin A supplementation. METHODS: The relationship between receipt of a vitamin A capsule and demographic and health indicators was examined in a cross-sectional study of 4013 children aged 12-59 months and their families who participated in the 2011 Nepal Demographic and Health Survey (NDHS), a nationally representative survey. Coverage of the vitamin A programme was compared with coverage estimates from surveys in 2001 and 2006. RESULTS: Coverage estimates of the national vitamin A programme for children aged 12-59 months as assessed by the 2001, 2006 and 2011 NDHS were 84.3%, 96.6% and 92.1%, respectively. Children who missed a vitamin A capsule were more likely to be younger and anaemic, have less educated parents, live in rural areas, and have higher child and infant mortality in the family. CONCLUSIONS: The national vitamin A supplementation programme in Nepal has relatively high coverage of children aged 12-59 months but still misses children in families with high child mortality. Further measures might be needed to sustain a high level of programme coverage.

Endocrine disrupting activities in sewage effluent and river water determined by chemical analysis and in vitro assay in the context of granular activated carbon upgrade.

As part of endocrine disruption in catchments (EDCAT) programme, this work aims to assess the temporal and spatial variations of endocrine disrupting chemicals (EDCs) in River Ray, before and after the commissioning of a full-scale granular activated carbon (GAC) plant at a sewage treatment works (STW). Through spot and passive sampling from effluent and river sites, estrogenic and anti-androgenic activities were determined by chemical analysis and in vitro bio-assay. A correlation was found between chemical analyses of the most potent estrogens (estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2)) and yeast estrogen screen (YES) measurement, both showing clearly a reduction in estrogenic activity after the commissioning of the GAC plant at the STW. During the study period, the annual average concentrations of E1, E2 and EE2 had decreased from 3.5 ng L(-1), 3.1 ng L(-1) and 0.5 ng L(-1) to below their limit of detection (LOD), respectively, with a concentration reduction of at least 91%, 81% and 60%. Annual mean estrogenic activity measured by YES of spot samples varied from 1.9 ng L(-1) to 0.4 ng L(-1) E2 equivalent between 2006 and 2008 representing a 79% reduction. Similarly, anti-androgenic activity measured by yeast anti-androgen screen (anti-YAS) of spot samples was reduced from 148.8 to 22.4 µg flutamide L(-1), or by 85%. YES and anti-YAS values were related to each other, suggesting co-existence of both types of activities from chemical mixtures in environmental samples. The findings confirm the effectiveness of a full-scale GAC in removing both estrogenic and anti-androgenic activities from sewage effluent.

Improved removal of estrogenic and pharmaceutical compounds in sewage effluent by full scale granular activated carbon: impact on receiving river water.

Sewage effluents are widely recognised as the main source of emerging contaminants, such as endocrine disrupting chemicals (EDCs) and pharmaceuticals in surface waters. A full-scale granular activated carbon (GAC) plant has been installed as an advanced technology for the removal of these contaminants, in a major sewage treatment works (STW) in South-West England as part of the UK National Demonstration Programme for EDCs. This study presented for the first time, an assessment of the impact of a recently commissioned, post-tertiary GAC plant in the removal of emerging contaminants in a working STW. Through regular sampling followed by solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), a significant reduction in the concentrations of steroidal estrogens was observed (>43-64%). In addition, significant reductions were observed for many of the pharmaceutical compounds such as mebeverine (84-99%), although the reduction was less dramatic for some of the more widely used pharmaceuticals analysed, including carbamazepine and propranolol (17-23%).

Sexual health of adults working in pornographic films.

We report the frequency of sexually transmitted infections (STIs) diagnosed in performers in the adult pornographic film industry. Over a 13 month period, 445 STI screens were performed in 115 patients, 56 women and 59 men. All reported unprotected sex during filming. Seventy-five percent (86) had at least one sexual partner outside work, and 90% used condoms inconsistently with them. Women worked exclusively with women (23%), men only (38%) or both genders (39%). Almost all men (97%) worked exclusively heterosexually. Thirty-eight percent (44/115) were diagnosed with 77 STIs, including non-specific urethritis (51), gonorrhoea (10), chlamydia (6) and genital warts (6). Gonorrhoea was found exclusively at the pharynx in three heterosexual men. There were no cases of HIV, syphilis, hepatitis B or hepatitis C. Monthly screening and certification is a working requirement for this population but STIs are common in an industry where unprotected sex is the norm.

Reduced glomerular filtration rate but sustained virologic response in HIV/hepatitis B co-infected individuals on long-term tenofovir.

Reports have described a decrease in glomerular filtration rate (eGFR) associated with tenofovir disoproxil fumarate (TDF) use in HIV positive individuals. However, no study has examined renal function over a prolonged period in HIV/hepatitis B virus (HBV) co-infected patients. We assessed the long-term durability and toxicity of TDF in a cohort of 39 e antigen (eAg) positive co-infected patients commenced on TDF 245 mg daily either in addition to or as part of standard antiretroviral therapy. Immunological and virological parameters were followed to 260 weeks, with the median follow-up period being 251 weeks (range 69-290 weeks). eGFR was calculated using the Modification in Diet in Renal Disease equation. On treatment at 260 weeks, 88% (14/16) had HIV viral load <50 copies/mL, median CD4 count rose from 318 to 532 cells/mm(3), median alanine aminotransferase (ALT) fell from 61 IU/L to 42 IU/L, with 35% (7/20) having a normal ALT, median HBV DNA fell from 69 x 10(6) copies/mL to 500 copies/mL, with 75% (12/16) having an undetectable HBV DNA level and 55% (6/11) becoming eAg negative. Of those with detectable HBV DNA, none had TDF resistance mutations. The eGFR declined by 22.19 mL/min/1.73 mm(2) from baseline (P = 0.023) over this period, which was unaffected by protease inhibitor use, baseline CD4 count, ALT or HBV DNA level. Three patients discontinued TDF therapy due to renal dysfunction. In conclusion, TDF has sustained efficacy but is associated with a significant decline in eGFR. Further larger studies are required to clarify this observation.

A comparison of three analytical techniques for the measurement of steroidal estrogens in environmental water samples.

Research into the analysis and monitoring of steroidal estrogens has grown significantly over the last decade, resulting in the emergence of a range of applicable techniques. In this study, three popular techniques, gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the analysis of three highly potent steroidal estrogens in the aquatic environment have been compared. It has been observed that overall, the three techniques appear comparable in generating similar estrogen concentrations for river and effluent samples. Of the three techniques, the GC-MS technique is the simplest to operate, but fails to detect the estrogens at the lower-end of environmentally relevant concentrations. The tandem MS techniques are more selective than MS, and therefore able to detect lower concentration levels of the three steroidal estrogens of interest. However, the LC-MS-MS technique is more susceptible to matrix interferences for the analysis of samples, resulting in a reduction of the signal-to-noise ratio and a subsequent reduction in reliability and stability compared to GC-MS-MS. With the GC-MS-MS technique offering increased selectivity, the lowest limits of detection, and no false positive identification, it is recommended to be the preferred analytical technique for routine analysis of estrogens in environmental water samples.

Pharmaceutical residues in wastewater treatment works effluents and their impact on receiving river water.

Various pharmaceutical residues are being discharged from wastewater treatment works (WTW) effluents, the impact of which on river water quality is of high relevance to environmental risk assessment. The concentrations of eleven pharmaceutical compounds were determined in three WTWs in England, and the river Ouse receiving effluents from Scaynes Hill WTW. Results show that five compounds propranolol, sulfamethoxazole, carbamazepine, indomethacin and diclofenac were detected in all wastewater and river water samples, with carbamazepine showing the highest concentrations (up to 2336 ng L(-1)) in WTW influent. Different compounds were removed to different extent in the WTWs, varying from 43 to 92%, with the highest performance obtained by the WTW with tertiary treatment (sand filtration). The pharmaceutical residues from Scaynes Hill WTW were eventually discharged into the river Ouse, causing an elevation in their concentrations downstream of the outfall. This was confirmed by the good agreement between measured concentrations and those predicted by a simple dilution model.

Is there a role for etravirine in patients with Nonnucleoside reverse transcriptase inhibitor resistance?

Etravirine is a next generation nonnucleoside reverse transcriptase inhibitor with activity against nonnucleoside reverse transcriptase inhibitor resistant HIV-1 virus. Susceptibility and virological response to etravirine is dependent on the type and number of nonnucleoside reverse transcriptase inhibitor resistance-associated mutations. We examined the predicted susceptibility of etravirine in patients experiencing virological failure secondary to nonnucleoside reverse transcriptase inhibitor resistance in our patient cohort.

Intravitreal steroids for macular edema in diabetes.

BACKGROUND: Macular edema is secondary to leakage from diseased retinal capillaries and is an important cause of poor central visual acuity in patients with diabetic retinopathy. OBJECTIVES: This review evaluated the effectiveness and safety of intraocular steroids in treating diabetic macular edema (DME). SEARCH STRATEGY: We searched CENTRAL, MEDLINE, EMBASE in June 2007, reference lists, Science Citation Index and conference proceedings. SELECTION CRITERIA: We included randomized clinical trials (RCTs) evaluating any form of intravitreal steroids for treating DME. DATA COLLECTION AND ANALYSIS: Two authors independently assessed eligibility, methodological quality and extracted data. We performed meta-analyses when appropriate. MAIN RESULTS: Seven studies, involving 632 DME eyes were included. Four examined the effectiveness of intravitreal triamcinolone acetate injection (IVTA), three examined intravitreal steroids implantation (fluocinolone acetonide implant (FAI) or dexamethasone drug delivery system (DDS)). Two trials were at low risk of bias, one was at median risk of bias, two were at high risk of bias and the remaining two were at unclear risk of bias. The preponderance of data suggest a beneficial effect from IVTA. Comparing IVTA with controls, the mean difference in visual acuity was -0.15 LogMAR (95% CI -0.21 to -0.09) at 3 months (based on three trials), -0.23 LogMAR (95% CI -0.33 to -0.13) at 6 months (two trials), -0.29 LogMAR (95% CI -0.47 to -0.11) at 9 months (one trial), and -0.11 LogMAR (95% CI -0.20 to -0.03) at 24 months (one trial), all in favor of IVTA. The relative risk (RR) for one or more lines improvement in visual acuity was 2.85 (95% CI 1.59 to 5.10) at 3 months (two trials), 1.25 (95% CI 0.66 to 2.38) at 6 months (one trial), and 2.17 (95% CI 1.15 to 4.11) at 24 months (one trial), all in favor of IVTA. We did not find evidence for three or more lines improvement in visual acuity. The mean difference in retinal thickness was -131.97 um (95% CI -169.08 to -94.86) at 3 months (two trials), -135.00 um (95% CI -194.50 to -75.50) at 6 months (one trial), -133.00 um (95% CI -199.86 to -66.14) at 9 months (one trial), and -59.00 um (95% CI -103.50 to -14.50) at 24 months (one trial), all in favor of IVTA. The RR for at least one grade macular edema resolution was 5.15 (95% CI 2.23 to 11.88) at 3 months in favor of IVTA (one trial). Two trials reported improved clinical outcome when FAI was compared to standard of care. Beneficial effect was also observed in one dexamethasone DDS trial. Increased intraocular pressure and cataract formation were side effects requiring monitoring and management. AUTHORS' CONCLUSIONS: RCTs included in this review suggest that steroids placed inside the eye by either intravitreal injection or surgical implantation may improve visual outcomes in eyes with persistent or refractory DME. Since the studies in our report focused on chronic or refractory DME, the question arises whether intravitreal steroids therapy could be of value in other stages of DME, especially the earlier stages either as standalone therapy or in combination with other therapies, such as laser photocoagulation.

What is the risk of mortality following diagnosis of multidrug-resistant HIV-1?

OBJECTIVES To estimate the risk of death and examine the predictors of death and virological/immunological response, following diagnosis of multidrug-resistant (MDR) HIV-1 in a UK multicentre cohort of HIV-infected individuals. METHODS Five hundred and seventy-two patients were identified with MDR HIV-1 between 1997 and 2004. Factors associated with survival and virological/immunological response 24-48 weeks after MDR diagnosis were determined by the Poisson and linear regression, respectively. RESULTS Patient characteristics: 86% males; median age 39 years; median CD4 and viral load (VL) at MDR diagnosis 230 cells/mm3 and 4.2 log10 copies/mL; median number of antiretroviral drugs previously exposed to 8. Sixty patients died over a median follow-up of 31 months (IQR: 17-50), giving an estimated mortality rate of 3.7 deaths per 100 person-years (95% CI 2.9-4.7) following MDR diagnosis. In adjusted analysis, higher CD4 count, lower VL, more recent calendar year, lower number of antiretroviral drugs previously exposed to and greater age at MDR diagnosis were associated with an increased chance of survival. There was some evidence of a better virological response at 24-48 weeks after MDR diagnosis in patients who changed regimen compared with patients who did not change regimen. CONCLUSIONS The risk of death following MDR diagnosis may be at least 3-fold the risk observed overall in HIV-infected individuals. Changing antiretroviral therapy following emergence of MDR HIV-1 may be associated with improved short-term virological response.

Cytomegalovirus viral load testing of blood using quantitative polymerase chain reaction in acutely unwell HIV-1-positive patients lacks diagnostic utility.

We examined the usefulness of measuring cytomegalovirus (CMV) viral load (VL) in blood using quantitative polymerase chain reaction (qPCR) in establishing a diagnosis of CMV end-organ disease in consecutive unwell HIV-infected patients. The indication for testing for CMV, CD4 count, CMV VL and presence of CMV end-organ disease were abstracted from case-notes. During a 42-month period, 216 tests were performed in 181 patients; the majority (61%) had CD4 counts <100 cells/microL. The prevalence of detectable CMV by qPCR was 43.5% (94/216) with a prevalence of CMV end-organ disease of 7.4% (16/216). Of patients with CMV detectable by qPCR, 72 % (50/69) had CD4 counts <100 cells/microL. For patients with definite CMV end-organ disease, the positive predictive value of detectable CMV by qPCR was 10% (9/94), and the negative predictive value was 98% (119/122). In acutely unwell HIV-infected patients, detection of CMV by qPCR is a poor predictor of CMV end-organ disease.

Rectal gonorrhoea in men -- is microscopy still a useful tool?

Rectal gonorrhoea (GC) in men may cause anal discharge or proctitis, but these symptoms have been shown to correlate poorly with rectal infection. Culture of Neisseria gonorrhoeae from an exposed site offers a readily available, sensitive and cheap diagnostic test, and is currently the gold standard for diagnosis; however, these results can take a few days and therefore do not offer an instant diagnosis. Gram staining of rectal smears for N. gonorrhoeae has a low sensitivity but a high specificity when performed by experienced personnel. We audited whether rectal microscopy increased the number of patients diagnosed and treated for rectal GC at initial presentation at one inner London genitourinary clinic over a 12-month period. One hundred and thirty-six episodes of rectal GC were identified in 132 men. In all, 134/136 had rectal microscopy of whom, 47/134 (35%) were smear-positive for GC. Of the 136 cases, 90 received antibiotics for GC at their first presentation. Twenty-four of 90 (27%) would not have been treated until culture results were available, if rectal microscopy had not been performed. These results suggest that rectal microscopy remains an important tool and increases the proportion of men treated for GC at their first attendance.

De novo hepatocellular carcinoma in a liver allograft associated with recurrent hepatitis B.

Recurrent hepatocellular carcinoma is well described following liver transplantation. However, de novo hepatocellular carcinoma in the allograft is rare. We describe the clinical and pathological features of a case of de novo hepatocellular carcinoma arising in a cirrhotic allograft 9 years following transplantation for chronic hepatitis B.

Herpes simplex virus infection of the central nervous system in human immunodeficiency virus-type 1-infected patients.

We report clinical, radiological and virological data from nine consecutive HIV-infected patients with herpes simplex virus (HSV) infection of the central nervous system (CNS). Three patients presented with confusion, two with fever and headache, two with anxiety and depression, one with slow mentation and memory loss and one with expressive dysphasia. Five patients had previous AIDS-defining diagnoses: four of these five patients had previous cutaneous HSV infection. HSV DNA was detected by the polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) in seven patients. HSV infection was diagnosed by brain biopsy (after negative PCR on CSF) in one patient and at autopsy in one patient (after negative CSF PCR and brain biopsy). Seven patients received specific anti-viral therapy; two died of unrelated causes and the other five recovered. Two patients were not treated, in one the diagnosis was made at autopsy and the other recovered spontaneously. HIV-infected patients with CNS HSV infection have a varied presentation. Diagnosis by PCR on CSF identified the majority of cases. With specific treatment the outcome was good.

Urinary catheter 'deflation cuff' formation: clinical audit and quantitative in vitro analysis.

OBJECTIVE: To investigate reports from district nursing staff of difficulty in removing long-term urinary catheters (LTCs) because of the formation of a 'cuff' on deflating the self-retaining balloon. PATIENTS AND METHODS: Problems experienced by district nurses when removing urethral and suprapubic LTCs were audited, noting the type of problem, the catheter and any action taken. Quantitative in vitro studies were conducted on the deflated self-retaining balloons after incubating a similar range of catheters in saline at 37 degrees C for 6 weeks, using suprapubic profilometry to assess the resistance to withdrawal (retention force). RESULTS: Questionnaires were returned on 154 patients with LTCs; 56% had urethral and 44% suprapubic catheters. The catheters were hydrogel-coated (83%), all-silicone (13%) and PTFE-coated (3%). Twenty-two (14%) of the sample reported problems with catheter removal in the previous year, including 15 (68%) with all-silicone catheters and 15 (68%) with suprapubic catheters; cuff formation was noted in 60%. In the laboratory, 10 of the balloons formed a 'cuff' on deflation, but there was great variability in the effect this had on the retention force, with values of 0.5-3 N for different catheters. CONCLUSIONS: Most problems with catheter removal involved all-silicone and suprapubic catheters. Suprapubic profilometry confirmed increased resistance to withdrawal by formation of a 'cuff' on deflation of the balloon of all-silicone catheters. These results suggest that the first choice of catheter material for long-term urethral and suprapubic use should be hydrogel-coated latex.

Sildenafil in the management of primary pulmonary hypertension.

Primary pulmonary hypertension is a rare disorder of unknown etiology with a poor prognosis. There is no cure, and drug therapy is effective in only a few patients. Calcium-channel antagonists and anticoagulants are the mainstay of therapy. Prostacyclin therapy leads to significant clinical improvement but its use is restricted due to high cost and complex drug delivery systems. Sildenafil is a selective vasodilator and has been shown to be effective in decreasing pulmonary vascular resistance in animal models of pulmonary hypertension. We report the use of sildenafil in two patients of primary pulmonary hypertension who were refractory to conventional drug therapy.

Experimental induction of equine protozoan myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease.

The effect of inoculation dose of Sarcocystis neurona sporocysts on the development of clinical neurologic disease in horses was investigated. Twenty-four seronegative weanling horses were subjected to the natural stress of transport and then randomly assigned to 6 treatment groups of 4 horses each. Horses were then immediately inoculated with either 10(2), 10(3), 10(4), 10(5), or 10(6) S. neurona sporocysts or placebo using nasogastric tube and housed indoors. Weekly neurologic examinations were performed by a blinded observer. Blood was collected weekly for antibody determination by Western blot analysis. Cerebrospinal fluid was collected before inoculation and before euthanasia for S. neurona antibody determination. Horses were killed and necropsied between 4 and 5 wk after inoculation. Differences were detected among dose groups based on seroconversion times, severity of clinical neurologic signs, and presence of microscopic lesions. Seroconversion of challenged horses was observed as early as 14 days postinfection in the 10(6) sporocyst dose group. Mild to moderate clinical signs of neurologic disease were produced in challenged horses from all groups, with the most consistent signs seen in the 10(6) sporocyst dose group. Histologic lesions suggestive of S. neurona infection were detected in 4 of the 20 horses fed sporocysts. Parasites were not detected in equine tissues by light microscopy, immunohistochemistry, or bioassay in gamma-interferon gene knockout mice. Control horses remained seronegative for the duration of the study and had no histologic evidence of protozoal infection.

An abdominal wall simulator for testing suprapubic urinary catheters.

Urinary catheters (drainage tubes) are in widespread use. The most common type of long-term catheter is the Foley, which is made from natural or synthetic rubber. Foley catheters are passed into the bladder via the urethra or the suprapubic puncture channel (through the abdominal wall). A simulator for the abdominal wall has been developed to simulate aspects of the interaction between it and a suprapubic catheter. The simulator is based on a slab of ultrasoft elastomer with tensionable reinforcing polyamide filaments. The behaviour of the simulator has been compared with data published. A soft membrane (contact pressure) transducer (SMT) was used and novel instrumented 'tongs' for lateral indentation of the puncture track giving indentation stiffness. Slab materials were used with shear moduli of 0.1 and 0.021 MPa. Two filament-tensioning methods were used: by clamping to a winding mechanism and by weights. The combination of the softer slab material and tensioning by weights gave good conformity to physiological data; other combinations did not.