Bone grafiting combined with Sauvé-Kapandji Procedures for the treatment of aseptic distal radius non-union.

Distal radius fractures are the most common type of upper limb fractures in adults. Non-union after distal radius fracture is rare, serious and unpredictable. The aim of our paper is to analyse the clinical and radiological outcomes of bone grafting and Sauvé-Kapandji Procedures for the treatment of aseptic distal radius non-union. We enrolled 13 patients with distal radius aseptic non-union. The following parameters were evaluated: The surgical time, elbow, forearm and wrist range of motion, the subjective quality of life and the wrist function measured by Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH), Pain Visual Analogic Score (VAS) and the complication rate. Bone union was measured using the radiographic union score as described by Radiographic Union Score (RUS). The evaluation endpoint was set at 24 months after surgery. All patients achieved fracture union. Grip strength improved by 12.4 kg. There was also improvement in wrist flexion, in wrist extension, and forearm pronosupination. These ranges of motion and grip strength improvements were statistically significant. Only 6 patients returned to full activity. This surgical technique represents a reliable alternative for treatment of distal radius aseptic non-unions. Further studies are needed to assess the long-term clinical results of this surgical procedure.

Reliability of S.A.R.A. (sterilization and reimplantation autograft) technique in long bone open fractures.

The reimplantation of small or large extruded bone segments is one of the most complex clinical management scenarios in the treatment of open fractures. No consensus exists regarding the efficiency of this technique. The aim of the study was to analyse the clinical and radiological outcomes of Sterilization and Reimplantation Autograft (S.A.R.A.) technique in open fractures. Therefore, fifteen skeletally mature patients with Gustilo-Anderson -IIIB type fractures treated with autograft reimplantation, were included in this study. The sample size was divided in two groups: patients with a loss of small segments (Group A - less than 5 cm) and those with large segments (Group B - greater than 5 cm). Eight patients belonged to Group A and seven to Group B. The treatment of contaminated bone may be performed by the following protocols: saline rinse, povidone-iodine scrub and saline rinse, retain periosteum, immersion in antibiotic solution (clindamycin and gentamicin and metronidazole), washing with physiological solution, acute reimplantation in Group A or reimplantation after 21 days in Group B after a bone freezing at -80°C. The Radiographic Union Score (RUS), pain visual analogic score (VAS), patient satisfaction and return to work were assessed at a mean follow-up of 24 months. No cases of superficial or deep infection were reported at 2-year follow-up. The fractures achieved a complete union in 14 patients; one patient belonging to Group A had a malabsorption of the replanted bone. Furthermore, povidone-iodine scrub, antibiotic solution immersion, and washing with physiological solution preserved the articular surface morphology. This study suggests that reimplantation of extruded short or long segments may represent a reliable alternative to amputation in open long bone fractures. Further studies are needed to define the most efficient technique for sterilizing the bone autograft to reduce the complication rate.

The challenge of non-union in subtrochanteric fractures with breakage of intramedullary nail: evaluation of outcomes in surgery revision with angled blade plate and allograft bone strut.

PURPOSE: Subtrochanteric fractures have a bimodal age distribution. They usually require open reduction and internal fixation. Closed reduction and intramedullary nail fixation rate are increased for this type of fracture. As a result, the hardware breakage and non-union rate is high among such patients. Our purpose is to evaluate the outcomes of the role of blade plate and bone strut allograft in the management of subtrochanteric non-union by femoral nailing. MATERIALS AND METHODS: We reported a group of 22 patients with subtrochanteric non-union, associated with breakage of the intramedullary nail with medial femoral allograft bone and lateral blade plate and wire (PS) s; and a group of 13 patients with subtrochanteric non-union, associated with breakage of the intramedullary nail treated with lateral blade plate and screws (CG). The chosen criteria to evaluate the two group during the clinical and radiological follow-up were the quality of life, measured by The Short Form (12) Health Survey (SF-12), the hip function and quality of life related to it, measured by the Harris Hip Score (HHS), bone healing, measured by Radiographic Union Score (RUS) by XR and CT at 1 year after the surgery, and postoperative complications. The evaluation endpoint was set at 12 months. RESULTS: The Bone healing measured by RUS occurred and also the full recovery before the first trauma measured by SF-12 and HHS are better in PS group. We only had three unimportant complications in PS while four breakage hardware in CG. CONCLUSION: We conclude that in complicated non-unions, the use of blade plate and bone strut allograft has a definite positive role in the management of such cases.

Percutaneous hallux valgus surgery: strengths and weakness in our clinical experience.

BACKGROUND AND AIM OF THE WORK: The Reverdin-Isham percutaneous osteotomy is indicated in the treatment of mild to moderate hallux valgus deformity. The aim of the work is the evaluation of the technique itself as a possible future landmark in the hallux valgus treatment. METHODS: Between January 2010 and January 2011 we have performed 138 percutaneous osteotomies. The patients were assessed with a clinical and radiological control after a median five months follow up. The score proposed by the American Orthopaedic Foot and Ankle Society was used for the clinical evaluation. RESULTS: The average score has improved from a preoperative median of 45 points to a postoperative median of 91 points. The technique has been largely accepted by the patients because of the speed of the procedure itself, the minimal invasiveness, the short pain and the immediate functional recovery. CONCLUSIONS: The results we have obtained with the Reverdin-Isham procedure have confirmed that this technique is a valid alternative to other percutaneous techniques and open surgical procedures. However the technique is not simple, it needs the strict indications observance and it needs a steep learning curve, those are features that impose further future studies.

The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation.

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

The RAD9-RAD1-HUS1 (9.1.1) complex interacts with WRN and is crucial to regulate its response to replication fork stalling.

The WRN protein belongs to the RecQ family of DNA helicases and is implicated in replication fork restart, but how its function is regulated remains unknown. We show that WRN interacts with the 9.1.1 complex, one of the central factors of the replication checkpoint. This interaction is mediated by the binding of the RAD1 subunit to the N-terminal region of WRN and is instrumental for WRN relocalization in nuclear foci and its phosphorylation in response to replication arrest. We also find that ATR-dependent WRN phosphorylation depends on TopBP1, which is recruited by the 9.1.1 complex in response to replication arrest. Finally, we provide evidence for a cooperation between WRN and 9.1.1 complex in preventing accumulation of DNA breakage and maintaining genome integrity at naturally occurring replication fork stalling sites. Taken together, our data unveil a novel functional interplay between WRN helicase and the replication checkpoint, contributing to shed light into the molecular mechanism underlying the response to replication fork arrest.

In vivo confocal microscopy in bilateral herpetic keratitis: a case report.

PURPOSE: Bilateral herpetic keratitis is a rare infection of the cornea. Ocular herpes in atopic patients is more often bilateral, with more frequent recurrences. The authors report a case of bilateral herpes simplex virus (HSV) keratitis during the active phase studied by in vivo confocal microscopy. METHODS: A 28-year-old man with 5 years history of unilateral HSV keratitis and atopic dermatitis was referred to the authors for a clinical and diagnostic evaluation.RESULTS. The corneas showed the typical features of dendritic HSV keratitis in both eyes. Examination by in vivo confocal microscopy demonstrated similar lesions in both eyes: a distortion of the superficial and basal epithelium and the presence of irregular hyperreflective structures and dendritic particles near the epithelial cells. The subbasal nerve plexus presented a tortuous appearance with hyperreflective areas and beadlike formations along the fibers. After a week of antiviral treatment, in vivo confocal microscopy examination demonstrated an irregular epithelium with highly reflective deposits and reflective areas. A reduction of nerve fiber bundles with a large number of beadlike formations and abnormal tortuosity was also noted. CONCLUSIONS: In vivo confocal microscopy enables a noninvasive evaluation of the ocular surface at a high magnification level. It could be useful for the early and differential diagnosis of corneal infections and when HSV keratitis recurrence is suspected.

Orbital metastasis associated with primary breast carcinoma in a man detected during peribulbar anesthesia for cataract surgery.

PURPOSE: A case of orbital infiltration by breast carcinoma in a male patient causing mild enophthalmos and subcutaneous mass detected accidentally during peribulbar anesthesia for cataract surgery is discussed. METHODS: The authors report a case of a 65-year-old man who came to the Ophthalmology Department for cataract surgery. During the presurgery peribulbar injection, a hard palpable mass located under the inferior left eyelid was noted, together with mild enophthalmos. A historical clinical screening revealed that 5 years previously the patient had undergone a right radical mastectomy to treat a ductal carcinoma of the breast. RESULTS: The patient underwent an incisional biopsy of the orbital mass that confirmed the clinical hypothesis of a metastasis. DISCUSSION: This case highlights the importance of the collection and screening of detailed clinical information on the patient before every ophthalmic operation including cataract surgery. The occurrence of progressive enophthalmos can represent an unusual symptom of orbital metastasis, commonly presenting with proptosis and diplopia.

Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair.

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Werner's syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle.

Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse.

Investigation of G2-phase chromosomal radiosensitivity in hereditary non-polyposis colorectal cancer cells.

PURPOSE: To investigate whether cells from hereditary nonpolyposis colorectal cancer (HNPCC) patients, a genetic condition characterized by constitutional mutations in DNA mismatch repair genes and associated with predisposition to colorectal carcinoma (CRC), could present a higher G2 chromosomal radiosensitivity. It is generally hypothesized that cancer predisposition in HNPCC is associated with the loss of the wild-type allele in somatic cells, resulting in defective DNA mismatch repair but, to date, no data on G2 radiosensitivity have been reported for HNPCC. MATERIALS AND METHODS: Lymphoblastoid cell lines derived from six HNPCC patients heterozygous for MLH1, one HNPCC patient carrying a mutant MSH2 allele and three healthy controls were treated with 50 cGy of X-rays and sampled at various harvesting times, monitoring cell-cycle progression by 5-bromo-2-deoxyuridine (BrdUrd) incorporation in order to analyse chromosomal damage in the homogeneous G2 population. RESULTS: There were no differences between lymphoblasts derived from patients in the frequency of G2 chromosomal aberrations induced by X-rays when compared with control cell lines. However, despite the absence of G2 radiosensitivity in HNPCC cells, lymphoblasts from patients heterozygous for MLH1 mutations showed a higher induction of chromatid exchanges. CONCLUSIONS: The observed possible incorrect rejoining of double-strand breaks in MLH1 heterozygotes would be an additional and important factor contributing to loss of heterozygosity in HNPCC patients.

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage.

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.

Evidence that camptothecin-induced aberrations in the G(2) phase of cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription.

It is widely accepted that camptothecin (CPT) is an S-dependent genotoxin. In this study, we aimed to elucidate the 'puzzling' induction of chromosomal damage by CPT in the G(2) phase of CHO cells, where no DNA synthesis is expected, focusing the attention on the possible role of the ongoing RNA synthesis, supposed to cause the conversion of CPT-single stranded cleavage complexes spaced closely on opposite DNA strands into DNA double strand breaks (DSB's) by the action of traversing RNA polymerase.CHO AA8 and its parental mutant EM9 cell lines were pre-treated with alpha-amanitin, which prevents transcription to pre-m-RNA and challenged cells with CPT for the last hour in culture to evaluate whether G(2)-CPT-induced aberrations would have been reduced or abolished in the absence of RNA synthesis compared with G(2)-CPT treatment alone. The results obtained indicated a marked and significant reduction of aberration yields, to almost the control values (alpha-amanitin alone) when inhibition of RNA synthesis was substantial (3h total alpha-amanitin). Partial inhibition of RNA synthesis (2h total alpha-amanitin) slightly reduced the CPT-induced aberrations yield only at the high dose-level employed of CPT (20mM). This finding strongly supports the hypothesis that CPT-single stranded cleavages complexes spaced closely on opposite DNA strands are converted into DNA double strand breaks by the action of traversing RNA polymerase.

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle.

PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle.

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.

Lack of effect of caffeine post-treatment on X-ray-induced chromosomal aberrations in Werner's syndrome lymphoblastoid cell lines: a preliminary report.

PURPOSE: To investigate whether in Werner's syndrome cells the G2 phase of the cell cycle has some abnormal response to post-treatment with agents such as caffeine and hydroxyurea known to interfere with cellular response to DNA damage. MATERIALS AND METHODS: Two Werner's syndrome lymphoblastoid cell lines (KO375 and DJG) and the normal cell line SNW646 were exposed to 50 cGy of X-rays or mitomycin-C and posttreated with caffeine or hydroxyurea in the G2 phase of the cell cycle. RESULTS: Hydroxyurea post-treatment potentiated the X-ray-induced aberration levels both in the normal and Werner's syndrome (KO375 and DJG) cell lines; in contrast caffeine was only effective in the normal cell line. Similar results were observed when Werner's syndrome cells were treated in the G1 phase with the S-dependent agent mitomycin-C and post-treated with caffeine in G2, extending the observation that Werner's syndrome cells are unaffected by caffeine G2 post-treatment. CONCLUSIONS: These results show a lack of caffeine effect in Werner's syndrome cells, suggesting an involvement of the Werner's syndrome protein in the signal transduction pathway by which caffeine could override the DNA damage induced G2 checkpoint.

Chromosome radiosensitivity in human G2 lymphocytes and cell-cycle progression.

PURPOSE: To investigate the possibility that the differential G2-phase radiosensitivity of human peripheral blood lymphocytes, found in normal individuals using the 'G2-phase chromosome radiosensitivity assay', could be attributed to heterogeneity in cellular progression to mitosis rather than differences in radiosensitivity. MATERIALS AND METHODS: Human peripheral blood lymphocytes, from four different donors, were exposed to 50 cGy X-rays and sampled at different times. The progression of cells into mitosis was monitored by 5-bromo 2'-deoxyuridine (BrdUrd) incorporation. RESULTS: The heterogeneous G2-phase chromosome radiosensitivity among different donors was abolished when homogeneous G2-phase cell populations were scored; they contained similar frequencies of cells in early or late G2-phase. CONCLUSIONS: The heterogeneous G2-phase chromosome radiosensitivity, usually found in different normal donors, is caused by the analysis of different cell populations rather than reflecting intrinsic differences in radiosensitivity.

Caffeine effect on the mitotic delay induced by G2 treatment with UVC or mitomycin C.

It is well established that DNA lesions trigger cell cycle check-points causing a mitotic delay that is required for their repair before cells enter the mitotic phase. Caffeine, in some cases, can remove this delay and consequently potentiates the yield of induced chromosome aberrations. The objective of this study was to test the effect of a G2 treatment with S-dependent agents (UV light and mitomycin C) on the cell kinetics of a G2 cell population and evaluate whether post-treatments with caffeine could modulate removal of the expected cell cycle delay. Cell kinetics were monitored by analysing the mitotic index (MI) values in combination with the 5-bromo-2'-deoxyuridine (BrdUrd) labelling technique. Chinese hamster fibroblast cultures (AA8) were treated in G2 phase of the cell cycle with 8 and 15 J/m2 UV light or 0.1 and 0.6 microgram/ml mitomycin C for 1.5 h. Post-treatments with caffeine were performed at dose levels and recovery times where the mitotic indices were substantially reduced. The results obtained showed that both UV light and mitomycin C induced a G2 arrest, as indicated by MI values and the absence of BrdUrd-labelled metaphases. For UV light the G2 block was observed at lower and higher dose levels after 1.5 h, while for mitomycin C it was observed only at the higher dose level after 1 h. However, in both cases the block lasted approximately 1 h, after which, even though slowed down, the cell population entered mitosis, as indicated by increased MI values. This block was not removed by caffeine post-treatment. In contrast, caffeine G2 post-treatment was able to remove G2 arrest induced by G1-S treatments. Accordingly, our results suggest that both UV light- and mitomycin C-induced damage must be processed during S phase to allow caffeine to remove induced G2 blocks.