Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein-1 and human transforming growth factor-ß3 in Class II furcation defects of Papio ursinus.

BACKGROUND AND OBJECTIVE: Binary applications of recombinant human osteogenic protein-1 (hOP-1) and transforming growth factor-ß3 (hTGF-ß3) synergize to induce pronounced bone formation. To induce periodontal tissue regeneration, binary applications of hOP-1 and hTGF-ß(3) were implanted in Class II furcation defects of the Chacma baboon, Papio ursinus. MATERIAL AND METHODS: Defects were created bilaterally in the furcation of the first and second mandibular molars of three adult baboons. Single applications of 25 µg hOP-1 and 75 µg hTGF-ß(3) in Matrigel(®) matrix were compared with 20:1 binary applications, i.e. 25 µg hOP-1 and 1.25 µg hTGF-ß(3). Morcellated fragments of autogenous rectus abdominis striated muscle were added to binary applications. Sixty days after implantation, the animals were killed and the operated tissues harvested en bloc. Undecalcified sections were studied by light microscopy, and regenerated tissue was assessed by measuring volume and height of newly formed alveolar bone and cementum. RESULTS: The hOP-1 and hTGF-ß(3) induced periodontal tissue regeneration and cementogenesis. Qualitative morphological analysis of binary applications showed clear evidence for considerable periodontal tissue regeneration. Quantitatively, the differences in the histomorphometric values did not reach statistical significance for the group size chosen for this primate study. The addition of morcellated muscle fragments did not enhance tissue regeneration. Binary applications showed rapid expansion of the newly formed bone against the root surfaces following fibrovascular tissue induction in the centre of the treated defects. CONCLUSION: Binary applications of hOP-1 and hTGF-ß(3) in Matrigel(®) matrix in Class II furcation defects of P. ursinus induced substantial periodontal tissue regeneration, which was tempered, however, by the anatomy of the furcation defect model, which does not allow for the rapid growth and expansion of the synergistic induction of bone formation, particularly when additionally treated with responding myoblastic stem cells.

Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR.

The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.

Possible importation and subsequent cross-transmission of OXA-48-producing Klebsiella pneumoniae, France, 2010.

We report the possible first patient-to-patient transmission of Klebsiella pneumoniae with decreased susceptibility to imipenem and producing OXA-48, CTX-M15, TEM-1 and OXA-1 in a French hospital.

Induction of cementogenesis and periodontal ligament regeneration by recombinant human transforming growth factor-beta3 in Matrigel with rectus abdominis responding cells.

BACKGROUND AND OBJECTIVE: In primates and in primates only, the transforming growth factor-b proteins induce endochondral bone formation. Transforming growth factor-b3 also induces periodontal tissue regeneration. Two regenerative treatments using human recombinant transforming growth factor-b3 were examined after implantation in mandibular furcation defects of the nonhuman primate, Papio ursinus. MATERIAL AND METHODS: Class III furcation defects were surgically created bilaterally in the mandibular first and second molars of two adult Chacma baboons (P. ursinus). Different doses of recombinant transforming growth factor-beta3 reconstituted with Matrigel matrix were implanted in the rectus abdominis muscle to induce heterotopic ossicles for subsequent transplantation to selected furcation defects. Twenty days after heterotopic implantation, periodontal defects were re-exposed, further debrided and implanted with minced fragments of induced heterotopic ossicles. Contralateral class III furcation defects were implanted directly with recombinant transforming growth factor-beta3 in Matrigel matrix with the addition of minced fragments of autogenous rectus abdominis muscle. Treated quadrants were not subjected to oral hygiene procedures so as to study the effect of the direct application of the recombinant morphogen in Matrigel on periodontal healing. Histomorphometric analyses on undecalcified sections cut from specimen blocks harvested on day 60 measured the area of newly formed alveolar bone and the coronal extension of the newly formed cementum along the exposed root surfaces. RESULTS: Morphometric analyses showed greater alveolar bone regeneration and cementogenesis in furcation defects implanted directly with 75 microg of transforming growth factor-beta3 in Matrigel matrix with the addition of minced muscle tissue. CONCLUSION: Matrigel matrix is an optimal delivery system for the osteogenic proteins of the transforming growth factor-beta superfamily, including the mammalian transforming growth factor-beta3 isoform. The addition of minced fragments of rectus abdominis muscle provides responding stem cells for further tissue induction and morphogenesis by the transforming growth factor-beta3 protein.

Cementogenesis and the induction of periodontal tissue regeneration by the osteogenic proteins of the transforming growth factor-beta superfamily.

The antiquity and severity of periodontal diseases are demonstrated by the hard evidence of alveolar bone loss in gnathic remains of the Pliocene/Pleistocene deposits of the Bloubank Valley at Sterkfontein, Swartkrans and Kromdrai in South Africa. Extant Homo has characterized and cloned a superfamily of proteins which include the bone morphogenetic proteins that regulate tooth morphogenesis at different stages of development as temporally and spatially connected events. The induction of cementogenesis, periodontal ligament and alveolar bone regeneration are regulated by the co-ordinated expression of bone morphogenetic proteins. Naturally derived and recombinant human bone morphogenetic proteins induce periodontal tissue regeneration in mammals. Morphological analyses on undecalcified sections cut at 3-6 mum on a series of mandibular molar Class II and III furcation defects induced in the non-human primate Papio ursinus show the induction of cementogenesis. Sharpey's fibers nucleate as a series of composite collagen bundles within the cementoid matrix in close relation to embedded cementocytes. Osteogenic protein-1 and bone morphogenetic protein-2 possess a structure-activity profile, as shown by the morphology of tissue regeneration, preferentially cementogenic and osteogenic, respectively. In Papio ursinus, transforming growth factor-beta(3) also induces cementogenesis, with Sharpey's fibers inserting into newly formed alveolar bone. Capillary sprouting and invasion determine the sequential insertion and alignment of individual collagenic bundles. The addition of responding stem cells prepared by finely mincing fragments of autogenous rectus abdominis muscle significantly enhances the induction of periodontal tissue regeneration when combined with transforming growth factor-beta(3) implanted in Class II and III furcation defects of Papio ursinus.

Effect of antibiotic therapy on human fecal microbiota and the relation to the development of Clostridium difficile.

The gastrointestinal tract is a complex ecosystem. Recent studies have shown that the human fecal microbiota is composed of a consortium of microorganism. It is known that antibiotic treatment alters the microbiota, facilitating the proliferation of opportunists that may occupy ecological niches previously unavailable to them. It is therefore important to characterize resident microbiota to evaluate its latent ability to permit the development of pathogens such as Clostridium difficile. Using samples from 260 subjects enrolled in a previously published clinical study on antibiotic-associated diarrhea, we investigated the possible relationship between the fecal dominant resident microbiota and the subsequent development of C. difficile. We used molecular profiling of bacterial 16S rDNA coupled with partial least square (PLS) regression analysis. Fecal samples were collected on day 0 (D0) before antibiotic treatment and on day 14 (D14) after the beginning of the treatment. Fecal DNA was isolated, and V6-to-V8 regions of the 16S rDNA were amplified by polymerase chain reaction with general primers and analyzed by temporal temperature gradient gel electrophoresis (TTGE). Main bacteria profiles were compared on the basis of similarity (Pearson correlation coefficient). The characteristics of the microbiota were determined using PLS discriminant analysis model. Eighty-seven TTGE profiles on D0 have been analyzed. The banding pattern was complex in all cases. The subsequent onset of C. difficile was not revealed by any clustering of TTGE profiles, but was explained up to 46% by the corresponding PLS model. Furthermore, 6 zones out of the 438 dispatched from the TTGE profiles by the software happened to be specific for the group of patients who acquired C. difficile. The first approach in the molecular phylogenetic analysis showed related sequences to uncultured clones. As for the 87 TTGE profiles on D14, no clustering could be found either, but the subsequent onset of C. difficile was explained up to 74.5% by the corresponding PLS model, thus corroborating the results found on D0. The non exhaustive data of the microbiota we found should be taken as the first step to assess the hypothesis of permissive microbiota. The PLS model was used successfully to predict C. difficile development. We found that important criteria in terms of main bacteria could be markedly considered as predisposing factors for C. difficile development. Yet, the resident microbiota in case of antibiotic-associated diarrhea has still to be analyzed. Furthermore, these findings suggest that strategies reinforcing the ability of the fecal microbiota to resist to modifications would be of clinical relevance.

[Totally implantable venous access ports: frequency of complications and analysis of bacterial contamination after ablation]. / Cathéters à chambre implantable: épidémiologie des complications et étude microbiologique des dispositifs après ablation.

UNLABELLED: Totally implantable venous access ports (TIVAP) are valuable medical devices for long-term intravenous treatment such as parenteral nutrition, cancer chemotherapy or antiviral therapy. Implantation and use of these devices are each associated with infectious or mechanical complications. AIMS OF THE STUDY: To determine the frequency of complications and to analyze bacterial contamination of different parts of TIVAP (tip, septum, internal lumen of the port). MATERIAL AND METHODS: Clinical charts of patients, which TIVAP was removed between April 20th to December 31st 2003, were retrospectively reviewed. Infectious complications (local and septicemic) and non-infectious complications (i.e. obstruction, thrombosis, drug extravasation...) were defined using clinical and/or microbiological criteria. Quantitative culture from different parts of the TIVAP was performed. RESULTS: One hundred and ten patients (age 57 +/- 14-years-old, 94.3% cancers) were included, corresponding to 57,018 catheter-days: 39.1% had one or more non-infectious complications (density incidence: 0.86 for 1000 catheter-days). Among the 49 complications, obstruction, thrombosis, extravasations and malposition accounted for 30.6%, 30.6% 4.1% and 6% of cases. Twenty-one patients (19.1%) had an infectious complication: 11 were local and 14 were systemic (density incidence 0.43 for 1000 catheter-days). Bacteria responsible for TIVAP-associated bacteraemia were coagulase negative staphylococci (N = 2), Staphylococcus aureus susceptible to methicilline (N = 3), micrococci (N = 1), corynebacteria (N = 1) or Gram-negative bacilli (N = 8). Comparison of quantitative culture of the different parts of TIVAP with a threshold at 10(3) CFU/ml showed that culture of tip, septum and port has a sensitivity of 47.6% 57.1% and 61.9 %, respectively and a specificity of 100% 92.1% and 92.1%, respectively for the diagnosis of TIVAP infection. CONCLUSION: Complications associated to TIVAP are frequent but incidence that we have reported is comparable with previous studies. Analysis of internal lumen of the port is the most sensitive method for the diagnosis of TIVAP-associated infections.

CdeA of Clostridium difficile, a new multidrug efflux transporter of the MATE family.

The cdeA gene, cloned from Clostridium difficile clinical strain 714 under the control of its natural promoter made Escherichia coli and Clostridium perfringens resistant to ethidium bromide and acriflavin but had no effect on the susceptibility of the hosts to the following antibiotics: norfloxacin, ciprofloxacin, gentamicin, erythromycin, tetracyclin, and chloramphenicol. However, it was responsible for fluoroquinolone resistance in E. coli when it was cloned under the control of the Plac promoter. Quantitative reverse transcriptase (RT)-PCR showed that growth of C. difficile clinical strain 253 in the presence of subinhibitory concentrations of ethidium bromide significantly increased the transcription of cdeA, but this was not observed with ciprofloxacin. The deduced protein was homologous to the protein sequences of known efflux pumps from the third cluster (the so-called DinF branch) of the multidrug and toxic compound extrusion (MATE) family. CdeA caused ethidium bromide energy-dependent efflux in whole cells of E. coli. Efflux activity was stimulated by addition of Na+ ions, suggesting that CdeA, like other pumps of the MATE family, is a Na+-coupled efflux pump. CdeA is the first multidrug efflux transporter identified in C. difficile.

Is manual counting of corneal endothelial cell density in eye banks still acceptable? The French experience.

AIM: To examine the differences in manual endothelial cell counting methods in French eye banks and to analyse whether these differences could explain some substantial discrepancies observed in endothelial cell density (ECD) for corneas made available for transplant. METHODS: A questionnaire was sent to the 22 eye banks asking for details of the technical features of the light microscopes used, the microscope calibration, strategy for cell counting, the technical staff, and the method of presenting endothelial data. RESULTS: All eye banks responded and 91% (20/22) used only manual counting methods, in real time, directly through a microscope, and 62 different technicians, with varying experience, were involved in such counting. Counting of cells within the borders of a grid that were in contact with two adjacent borders was the most common method (17/22, 77%). Of the eight banks (8/22, 36%) that did not calibrate their microscopes, six reported the highest ECD values. Of the 14 others (64%), six applied a "magnification correcting factor" to the initial cell counts. In five of these cases, the corrected ECD was lower than estimated on initial count. Most of the banks (12/22, 55%) counted 100 cells or less in one to six non-adjacent zones of the mosaic. 14 of the banks (14/22, 64%) also graded cell polymegethism while seven (7/22, 32%) also graded pleomorphism ("hexagonality"). CONCLUSIONS: Lack of microscope calibration appears to be the leading cause of variance in ECD estimates in French eye banks. Other factors such as differences in counting strategy, the evaluation of smaller numbers of cells, and the different extent of experience of the technicians may also contribute to intraobserver and interobserver variability. Further comparative studies, including cross checking and the outcome of repeated counts from manual methods, are clearly needed with cross calibration to a computer based image archiving and analysis system.

A European survey of diagnostic methods and testing protocols for Clostridium difficile.

OBJECTIVE: To conduct a survey of the methods used in clinical microbiology laboratories in Europe to diagnose infection with Clostridium difficile. METHODS: A questionnaire was devised and sent to a co-ordinating member of the Study Group in each of eight European countries. This co-ordinator was in charge of forwarding the questionnaire to hospital laboratories arbitrarily selected. The number of laboratories in each country was determined on the basis of one laboratory for 10,000 beds of hospitalization. This questionnaire covered different aspects pertaining to Clostridium difficile associated to diarrhea (CDAD) diagnosis such as circumstances of request, criteria used for undertaking C. difficile investigations, methods used for the diagnosis, etc. RESULTS: A total of 212 questionnaires were completed and submitted for analysis: 87.7% of laboratories reported routinely performing C. difficile diagnostic tests. Methods used included toxin detection (93%), culture (55%), and glutamate dehydrogenase (GDH) detection (5.9%). Among the laboratories detecting toxins, different enzyme immunoassays (EIA) and cytotoxicity assays were used in 79% and 17.3% of cases, respectively. Among the different strategies reported, 4.8% were considered suboptimal for the diagnosis of C. difficile infections, but marked discrepancies could be observed between countries. The overall incidence (median) of CDAD was estimated at 1.1 for 1,000 patient admissions. CONCLUSION: The results of this study suggest marked discrepancies between laboratories and also between countries regarding the criteria by which C. difficile is investigated for, and the methods and the strategies that are used for the diagnosis of C. difficile. These discrepancies could be explained by the lack of clear guidelines for C. difficile diagnosis in each country, and by the importance that physicians attach to C. difficile. Precise guidelines for C. difficile diagnosis would be the first step to make possible accurate comparison of the incidence and the epidemiology of CDAD from one hospital to another or from one country to another.

[Complications due to peripheral venous catheterization. Prospective study]. / Complications liées au cathétérisme veineux périphérique. Etude prospective.

OBJECTIVE: Peripheral venous catheter (PVC)-associated complications were prospectively evaluated in a 2 month-study performed in 3 different wards. METHODS: For each inserted PVC, the following complications were observed daily by an external investigator: tenderness, erythema, swelling or induration, palpable cord and purulence. PVC that were removed were systematically sent to the Microbiology department and analysed according to the semi-quantitative method described by Brun-Buisson et al. RESULTS: A total of 525 PVC (corresponding to 1,036 catheterisation-days) were included. Main clinical complications were erythema (22.1%), tenderness (21.9%), swelling or induration (20.9%), palpable cord (2.7%) and purulence (0.2%). Phlebitis, defined by 2 or more of the following signs: tenderness, erythema, swelling or induration and palpable cord, was observed in 22%. Catheter colonization (> or = 103 CFU/ml) occurred in 13%. Bacteria isolated from colonized catheters were coagulase-negative staphylococci (88.1%), Staphylococcus aureus (7.1%) and Candida sp. (4.8%). Multivariate risk factor analysis showed that age > or = 55 y. (OR = 3.16, p = 0.003), insertion on articulation site (OR = 2.94, p = 0.01) or in jugular vein (OR = 8.18, p = 0.01) and > 72 hour-catheterisation (OR = 4.74, p = 0.0003) were significantly associated with PVC colonization. Risk factors for phlebitis were skin lesions (OR = 1.88, p < 0.016), active infection unrelated to PVC (OR = 2.8, p = 0.001), "poor quality" peripheral vein (OR = 2.46, p < 0.02) and > 72 hour-catherisation (OR = 2.38, p = 0.009). CONCLUSION: Complications associated with peripheral venous catheters are frequent but remain benign. They could probably be reduced by a systematic change every 72-96 hours as recommended by different guidelines.

Antibiotic-associated diarrhoea and Clostridium difficile in the community.

BACKGROUND: Clostridium difficile is the main cause of nosocomial infectious diarrhoea and the causative agent of antibiotic-associated colitis. The involvement of C. difficile infection in antibiotic-associated diarrhoea in the community is poorly documented. METHODS: We studied prospectively 266 adult out-patients in the Paris (France) area who were prescribed a 5-10-day course of antimicrobial chemotherapy. Stools were screened for C. difficile before and 14 days after the start of treatment by standard culture, toxigenic culture and testing for the cytopathic effect of toxin B. Patients were requested to note daily stool frequency and consistency. Diarrhoea was defined as the passage of at least three loose stools per day. RESULTS: Forty-six (17.5%) of the 262 assessable patients had diarrhoea during the study period. Diarrhoea was mild and self-limited in all patients, and lasted for only 1 day in 65.6% of cases. C. difficile was isolated before and after treatment from one patient, who did not develop diarrhoea. C. difficile was detected only on day 14 in 10 patients (3.8%). The isolate was toxin producing in seven patients. Four of these seven patients had mild self-limited diarrhoea. Toxin-producing C. difficile was isolated significantly more frequently from patients who had diarrhoea than from those who were diarrhoea free (8.7% vs. 1.4%, P = 0.02). CONCLUSION: The acquisition of toxin-producing C. difficile appears to be frequent during antimicrobial chemotherapy in the community [estimated rate of 2700 (1150-5400) cases per 100 000 exposures to antibiotics]. However, C. difficile is not the main agent of mild antibiotic-associated diarrhoea in out-patients.

[Cornea donation consent by telephone]. / Recueil du consentement au don de cornée par téléphone.

BACKGROUND: The cornea donation process often runs into problems of obtaining family consent. A face-to-face interview is often not possible for logistical reasons. We carried out a prospective study on the effectiveness of telephone contact in obtaining donation consent. MATERIAL AND METHODS: Consent was obtained by a single, non medical, hospital coordinator. He contacted families selected on good staff-family relations during the patient's stay. If a face-to-face interview was not possible, a telephone interview was conducted using a standardized procedure. RESULTS: Over 21 months, 334 families were contacted, either in a face-to-face interview (142, 42.5%) or by telephone (192, 57.5%). Donation consent was obtained in 66.5% of cases, 106 times by telephone (47.7%) and 116 times in the face-to-face interview (52.3%). The acceptance rate was 55.2% by telephone and 81.6% face to face (p<0.001). CONCLUSIONS: The telephone interview was an effective method for obtaining consent for cornea donation. Although the acceptance rate using this method is lower than the face-to-face interview, using the telephone should not be overlooked as this enabled procurement of nearly half the corneas in our hospital.

[Automated analyser of organ cultured corneal endothelial mosaic]. / Mise au point d'un analyseur automatique de l'endothélium des greffons cornéens conservés.

PURPOSE: Until now, organ-cultured corneal endothelial mosaic has been assessed in France by cell counting using a calibrated graticule, or by drawing cells on a computerized image. The former method is unsatisfactory because it is characterized by a lack of objective evaluation of the cell surface and hexagonality and it requires an experienced technician. The latter method is time-consuming and requires careful attention. We aimed to make an efficient, fast and easy to use, automated digital analyzer of video images of the corneal endothelium. METHODS: The hardware included a PC Pentium III ((R)) 800 MHz-Ram 256, a Data Translation 3155 acquisition card, a Sony SC 75 CE CCD camera, and a 22-inch screen. Special functions for automated cell boundary determination consisted of Plug-in programs included in the ImageTool software. Calibration was performed using a calibrated micrometer. Cell densities of 40 organ-cultured corneas measured by both manual and automated counting were compared using parametric tests (Student's t test for paired variables and the Pearson correlation coefficient). RESULTS: All steps were considered more ergonomic i.e., endothelial image capture, image selection, thresholding of multiple areas of interest, automated cell count, automated detection of errors in cell boundary drawing, presentation of the results in an HTML file including the number of counted cells, cell density, coefficient of variation of cell area, cell surface histogram and cell hexagonality. The device was efficient because the global process lasted on average 7 minutes and did not require an experienced technician. The correlation between cell densities obtained with both methods was high (r=+0.84, p<0.001). The results showed an under-estimation using manual counting (2191+/-322 vs. 2273+/-457 cell/mm(2), p=0.046), compared with the automated method. CONCLUSIONS: Our automated endothelial cell analyzer is efficient and gives reliable results quickly and easily. A multicentric validation would allow us to standardize cell counts among cornea banks in our country.

Automated tri-image analysis of stored corneal endothelium.

BACKGROUND: Endothelial examination of organ culture stored corneas is usually done manually and on several mosaic zones. Some banks use an image analyser that takes account of only one zone. This method is restricted by image quality, and may be inaccurate if endothelial cell density (ECD) within the mosaic is not homogeneous. The authors have developed an analyser that has tools for automatic error detection and correction, and can measure ECD and perform morphometry on multiple zones of three images of the endothelial mosaic. METHODS: 60 human corneas were divided into two equal groups: group 1 with homogeneous mosaics, group 2 with heterogeneous ones. Three standard microscopy video images of the endothelium, graded by quality, were analysed either in isolation (so called mono-image analysis) or simultaneously (so called tri-image analysis), with 50 or 300 endothelial cells (ECs) counted. The automated analysis was compared with the manual analysis, which concerned 10 non-adjacent zones and about 300 cells. For each analysis method, failures and durations were studied according to image quality. RESULTS: All corneas were able to undergo analysis, in about 2 or 7.5 minutes for 50 and 300 ECs respectively. The tri-image analysis did not increase analysis time and never failed, even with mediocre images. The tri-image analysis of 300 ECs was always most highly correlated with the manual count, particularly in the heterogeneous cornea group (r=0.94, p<0.001) and prevented serious count errors. CONCLUSIONS: This analyser allows reliable and rapid analysis of ECD, even for heterogeneous endothelia mosaics and mediocre images.

[Corneal harvesting from donors over 85 years of age: cornea outcome after banking and grafting]. / Prélèvements de cornées après 85 ans: issue des greffons après organoculture et devenir des greffés.

PURPOSE: To study the suitability of corneas from very old donors for graft after organ culture and their clinical and endothelial outcomes in recipients after perforating keratoplasty. METHODS: We stored 419 corneas at 31 degrees C for 13.1 +/- 4 days (mean +/- SD) and then divided them according to donor age: group 1, donors under 85 years of age (n=3 3 0, 79%, 16-84 years old), and group 2, donors over the age of 85 (n=8 9, 21%, 85-100 years old). Endothelial density at the time of harvest and before and after organ culture, rates of suitability for grafting, and clinical and endothelial outcomes of the 196 keratoplasty procedures were compared in a prospective longitudinal study of the 2 groups, with a mean follow-up of 25 months. The corneas were grafted with no pre-established policy on matching with the age of the receiver. Statistical analysis was carried out on SPSS 10.0: Chi(2), Student t test, and Kaplan Meier survival curves. RESULTS: The average age of the donors was 72.1 +/- 16.7 years. The macroscopic aspect of the corneas was judged to be of slightly lower quality in group 2. No statistically significant difference was found in overall suitability for transplantation (group 1, 45% vs group 2, 54%, p=0.17) but elimination for low endothelial density was more frequent in group 2 (67% vs 39%, p=0.001). Cell density at the beginning of organ culture was lower in very old corneas than in younger corneas (respectively, 2116 +/- 368 vs 2 311 +/- 360 cell/mm(2), p=0.002) but no difference was apparent at the end of organ culture (respectively, 2 011 +/- 285 vs 2 090 +/- 296, p=0.12) because very old corneas lost fewer cells than younger ones (respectively, 5.6% vs 10.0%, p=0.001). There was no correlation between donor/receiver age (r=0. 337) but group 1 corneas were slightly more frequently allotted to receivers with normal endothelium (p=0.019). During surgery, the two groups did not differ in terms of the macroscopic aspect of the grafts. In the 196 grafted patients, and without age-matching, overall graft survival (86% vs 79%, p=0.275), visual acuity, and endothelial density (1 194 +/- 469 vs 1098 +/- 545 cells/mm(2), p=0.387) did not differ at the completion of the study. DISCUSSION: The corneas from very old donors were macroscopically of poorer quality and had a lower cellular endothelial density at harvesting, but these differences disappeared after organ culture because of greater cell loss in corneas from younger donors. Selection by organ culture ensures that functional, anatomical, and cellular results are not influenced by very old donor age. CONCLUSION: Considering the aging population in countries with a high standard of living, the techniques available for selecting corneas based on endothelial quality, and the increasing need for corneal grafts, the very old age should not be deemed off-limits for corneal harvesting.

Cornea procurement from very old donors: post organ culture cornea outcome and recipient graft outcome.

AIM: To study the suitability of corneas from very old donors for graft after banking and their clinical and endothelial outcomes in recipients. METHODS: 419 corneas stored in organ culture were divided into group 1, donors under 85 years (330 corneas) and group 2, "very old" donors aged 85 years and over (89 corneas). Endothelial cell density (ECD) before and after organ culture, discard rate before and after storage, and clinical and endothelial outcomes of the 196 penetrating keratoplasties (PKP) (158 in group 1 and 38 in group 2) were compared in a prospective longitudinal study. RESULTS: Initial ECD was lower in group 2 than in group 1 and elimination for low ECD was more frequent in group 2 (respectively 38% v 20.2%, p=0.001). At the end of storage, because very old corneas lost fewer ECs than younger ones (respectively 4.2% v 9.5%, p=0.022), ECD was comparable between the two groups. The corneas of very old donors had a poorer macroscopic appearance at procurement and during surgery. Despite this, in grafted patients, overall graft survival in groups 1 and 2 (respectively 87.4% v 80.6%, p=0.197), visual acuity, and ECD did not differ at completion of the study (mean follow up 25 months). CONCLUSION: This study suggests that endothelial cell count during banking ensures that functional and cellular results of PKPs are not dramatically influenced by very old donor age. Considering Europe's ageing population, the very elderly should not be deemed off limits for corneal procurement.

A new crystalline phase of nitric acid dihydrate.

The crystal structure of a new high-temperature phase of nitric acid dihydrate, HNO(3).2H(2)O, has been determined at 225 K by single-crystal X-ray diffraction. The H atom of the nitric acid is delocalized to one water molecule, leading to an association of equimolar NO(3)(-) and H(5)O(2)(+) ionic groups. The asymmetric unit contains two molecules of HNO(3).2H(2)O. The two independent molecules are related by a pseudo-twofold c axis, by a translation of 0.54 (approximately (1/2)) along b, with a mean atomic distance difference of 0.3 A, except for one H atom of the water molecules (1.5 A), because of their different orientations in the two molecules. The two independent molecules, linked by strong hydrogen bonds, are arranged in layers. These layers are linked by weaker hydrogen bonds oriented approximately along the c axis. A three-dimensional hydrogen-bond network is observed.