Pathologic complete response (pCR) after neoadjuvant chemotherapy for breast cancer is associated with improved prognosis in aggressive tumor subtypes, including ERBB2- positive tumors. Recent adoption of pCR as a surrogate endpoint for clinical trials in early stage breast cancer in the neoadjuvant setting highlights the need for biomarkers that, alone or in combination, help predict the likelihood of response to treatment.MET, TOP2A, PTEN, and PIK3CA genes, each paired with centromeric probes to their respective chromosomes . Ki-67 expression was assessed by immunohistochemistry (IHC). Various parameters describing copy number alterations were evaluated for each gene and centromere probe to identify the optimal parameters for clinical relevance. Combinations of ISH parameters and IHC expression for Ki-67 were also evaluated.Biopsy specimens from 29 patients with invasive ductal carcinoma treated with trastuzumab-based therapy prior to definitive resection and pathologic staging were evaluated by dual color bright field in situ hybridization  using probes for MET gain or loss relative to chromosome 7  and gain of PTEN . Ki-67 expression was also found to associate significantly with pCR . Combining gain or loss of MET relative to chromosome 7 with Ki-67 expression further improved the association with pCR .Of the four genes and their respective chromosomes evaluated by ISH, two gene copy number parameters provided statistically significant associations with pCR: MET relative copy number and Ki-67 expression generated by dual ISH and IHC may help predict pCR in ERBB2-positive breast cancer treated with neoadjuvant chemotherapy and trastuzumab. These findings require validation in additional patient cohorts.An immunogenotypic signature of low complexity comprising The online version of this article (doi:10.1186/s12885-016-2743-x) contains supplementary material, which is available to authorized users. The propCR data . Given tPIK3CA and benefit from ERBB2-targeted therapy [PIK3CA mutations. In the NeoALLTO neoadjuvant trial which incorporated lapatinib as well as trastuzumab, patients with PIK3CA mutations were less likely to have a pCR, but there were no significant differences in progression-free or overall survival [PIK3CA gene copy number alterations and clinical outcomes in breast cancer, irrespective of ERBB2 status [PIK3CA alleles appears to contribute to resistance to PI3K inhibitors in preclinical breast cancer models [Genetic alterations in the phosphatidylinositol 3-kinase (PI3K)/V-AKT murine thymoma viral oncogene homolog (AKT)/mechanistic target of rapamycin (MTOR) pathway are common events in breast cancer , 5. Prec therapy –14. In t therapy  and NSAB therapy , there wsurvival . Other nsurvival , 14. Com2 status . Amplifir models .PTEN copy number alterations in response to ERBB2-targeted therapy.Phosphatase and tensin homolog (PTEN) is the 3’ lipid phosphatase for phosphatidylinositol-3,4,5-triphospate (PIP3), thereby negatively regulating downstream signaling by PIP3 after phosphorylation by PI3K , 18. PatERBB2 genes are located close to each other on the long arm of chromosome 17 and may be co-amplified in breast cancer [TOP2A gene amplification [TOP2A copy number have mainly been associated with response to anthracycline-based chemotherapy [TOP2A amplification or deletion in response to ERBB2-targeting has not been thoroughly investigated.The topoisomerase II alpha (TOP2A) and t cancer –29. Apprfication , 31 and fication , 32. Altotherapy , 33. TheMET copy numbers correlate with trastuzumab therapy failure in ERBB2-positive breast cancer [MET amplification or deletion in the response to adjuvant or neoadjuvant therapy for ERBB2-positive breast cancer is not well established.Overexpression of MET proto-oncogene, receptor tyrosine kinase (MET) occurs in 20 % – 30 % of invasive breast cancers  and is at cancer  and clint cancer . The sigPIK3CA, PTEN, TOP2A and MET, and their respective chromosomes, and expression of Ki-67 in a series of patients with ERBB2-positive tumors who were treated with chemotherapy and trastuzumab in the neoadjuvant setting. Various parameters representing gene and chromosome copy numbers were evaluated for association with pCR in an effort to identify parameters most effective for improving prediction in the neoadjuvant treatment of ERBB2-positive breast cancer.In this exploratory study using in situ hybridization (ISH) and immunohistochemistry (IHC), we assessed alterations in gene copy number for ERBB2 status by ISH, post-treatment pathologic stage, and presence or absence of a pCR were recorded for all patients . Of the 234 cases, 29 satisfied inclusion criteria which included a diagnosis of primary invasive breast cancer, neoadjuvant trastuzumab therapy, and a pre-treatment biopsy performed at the Cleveland Clinic. Pathology data was obtained from the Anatomic Pathology information system CoPath Plus . Clinical data was obtained from the electronic medical record Epic . The age, tumor size, pre-treatment clinical stage, hormone receptor status by IHC, Formalin-fixed paraffin-embedded (FFPE) needle biopsy specimens from early stage breast cancer patients treated in the neoadjuvant setting were obtained from the archives of the Cleveland Clinic . Chart review and study analyses were approved by the Cleveland Clinic institutional review board. Eligibility criteria for further evaluation included histologic confirmation of clinical stage IIA to IIIB, ERBB2 amplification by in fluorescent in situ hybridization (FISH), and neoadjuvant treatment that included trastuzumab. Post-treatment pathologic staging was obtained from pathology reports and confirmed by histologic evaluation. For this study, classification as pCR required the absence of any detectable invasive carcinoma in the breast specimen and the axillary lymph nodes .With respect to treatment, of the 29 patients in the cohort 11 were treated with anthracycline-based chemotherapy including cyclophosphamide, a taxane, and trastuzumab (i.e. ACTH) and 18 received a taxane and trastuzumab with or without carboplatin (TCH). Of the 15 patients with a pCR, 5 were treated with ACTH and 10 were given TCH. Of the 14 patients who did not have a pCR, 6 received ACTH and 8 were treated with TCH. A total of 12 patients presented with clinical stage IIA or IIB disease and 8 of these patients had a pCR. Of the 8 patients with a pCR, 3 were treated with ACTH and 5 were given TCH. Of the 4 stage II patients who did not have a pCR, 1 was treated with ACTH and 3 were given ACTH. In this small exploratory study there may be some imbalance in the distribution of stage II patients in the two treatment groups. However, we believe the distribution of patients treated with ACTH versus TCH is relatively well-balanced among those who did or did not have a pCR.Ki-67 automated IHC was performed on 3–6 μm thick sections of FFPE specimen blocks using primary antibody 30-9 with iVIEW detection on the VENTANA BenchMark XT automated stainer instrument  using the company recommended protocols.MET), and a centromere (CEN) probe for the chromosome on which the gene locus lies (e.g. CEN7): MET + CEN7, TOP2A + CEN17, PTEN + CEN10, and PIK3CA + CEN3. All gene probes were detected using peroxidase-catalyzed silver staining (SISH) and centromeric probes were detected using chromosome staining with alkaline phosphatase-catalyzed fast red staining (CISH). ISH probes and associated detection reagents are commercially available from Ventana Medical Systems.Automated in situ hybridization (ISH) was performed on 3–6 μm thick sections of FFPE specimen blocks using the Ventana Medical Systems dual ISH procedure  on the BenchMark XT automated stainer. Probes were hybridized in the following pairs, each comprising a gene locus probe, referred to by the name of a gene contained within the targeted region . Consistent with the timeframe in which the patients were treated, ERBB2 scoring methods were applied to FISH samples in accordance with the 2007 ASCOCAP guidelines [ERBB2CEP17 < 1.8), equivocal (ERBB2CEP17 1.8–2.2), or amplified (ERBB2CEP17 > 2.2). For the cases in which it was performed, ERBB2 IHC  was scored according to the 2007 ASCOCAP guidelines [idelines . Brieflyidelines  as 0, 1,MET/cell) or centromere/cell (e.g. CEN7/cell),the average number of gene or centromere copies per cell, designated as gene/cell (e.g. MET gain) or centromere gain (e.g. CEN7 gain),the percentage of cells with greater than 2 gene or centromere signals, designated as gene gain (e.g. MET loss) or centromere loss (CEN7 loss),the percentage of cells with less than 2 gene or centromere signals, designated as gene loss (e.g. MET gain or loss), or centromere gain or loss (e.g. CEN7 gain or loss),the percentage of cells with either greater than 2 or less than 2 gene locus or centromere signals, designated as gene gain or loss (e.g. MET/CEN7),the average number of gene copies per corresponding centromere copies, designated as gene locus/centromere (e.g. MET/CEN7 gain),the percentage of cells with more copies of the gene than the corresponding centromere, designated as gene/centromere gain (e.g. MET/CEN7 loss), andthe percentage of cells with fewer copies of the gene than the corresponding centromere, designated as gene/centromere loss (e.g. MET/CEN7 gain or loss).the percentage of cells with either more copies of the gene than the corresponding centromere or fewer copies of the gene than the corresponding centromere, designated as gene/centromere gain or loss  gene locus signals and CISH (red) centromere signals on a cell-by-cell basis. Only invasive carcinoma tumor cells were selected for cell-by-cell signal enumeration using a 40X objective in combination with 10X eyepieces. In general, 50 invasive tumor cells were enumerated per specimen except in several specimens for which fewer than 50 cells with good hybridization signals could be found . Gene locus and centromere copy number statuses were assessed using a number of different parameters, including:The average number of genes or centromeres per cell was calculated by summing the number of gene or centromere signals over all the cells enumerated within a specimen, and dividing by the number of cells enumerated. The average number of genes per centromere was calculated by summing the number of gene signals over all the cells enumerated and dividing by the sum of the centromere signals in all of the cells enumerated.For Ki-67 IHC staining interpretation was performed only for invasive tumor cells and was evaluated by selecting the area of invasive carcinoma with the highest proliferation rate and then determining the percentage of approximately 50 invasive tumor cell nuclei that were positive for Ki-67 expression. The cutoff for Ki-67 positively staining cells was determined empirically to provide the best combined sensitivity and specificity for pCR in this neoadjuvant setting. An additional data file (.xls) lists the values for each ISH and IHC parameter described above for each patient in the study [Additional file p-value.For each parameter a range of cutoffs was evaluated such that specimens with the parameter value greater than or equal to a cutoff were considered ‘high’ for that parameter and specimens with the parameter value less than the cutoff were considered ‘low’ for that parameter. Sensitivities and specificities for detecting patients with pCR, based on either the high parameter being positive for pCR or based on the low parameter being positive for pCR, were calculated for each parameter and each cutoff. Receiver Operating Characteristics (ROC) curves were generated as sensitivity versus 1 - specificity over all cutoffs tested, and Area Under the Curve (AUC) was calculated for each curve as one measure of a parameter’s ability to distinguish patients with pCR from patients without pCR, with AUC = 1 being ideal and progressively lower values being less favorable. AUC values near 0.5 indicate no ability to distinguish between patients. In addition to ROC analysis, 2X2 contingency tables were evaluated at each cutoff and probabilities from Fischer’s Exact test were used to gauge the statistical significance of the association between the binarized parameter  and pCR. The optimal cutoff for a parameter was the cutoff value providing the best combined sensitivity and specificity, which typically provided the lowest MET/CEN7 gain or loss with Ki-67 expression was generated by using the cutoff providing optimal sensitivity and specificity for MET/CEN7 gain or loss while varying the cutoff for Ki-67 over a wide range, with the combined parameters considered positive if both parameters were equal to or greater than the respective cutoff values, as described by Shultz, 1995 [In addition to the various single parameters, a ROC curve for the combinations of tz, 1995 .MET and CEN7 ISH counts, 25 had PTEN and CEN10 ISH counts, and 24 had both Ki-67 expression data and MET and CEN7 ISH counts. The mean and median age at presentation was 53 and 52 years, respectively. The mean and median pre-treatment tumor size was 49 and 41 mm, respectively (range = 12−86). Patients who presented with Stage IV disease and who underwent breast surgery were excluded. Overall, 15 of 29 (52 %) patients had a pCR, defined as ypT0/ypTis N0. Among the 14 patients who did not have a pCR, the residual invasive tumor measured less than 1 mm in 1 patient and 1 patient had residual ductal carcinoma in situ (DCIS) and a positive lymph node (ypTisN1).A total of 29 patients meeting inclusion criteria with tissue available for IHC and ISH studies were identified Table . Of thesTOP2A, MET, PTEN, and PIK3CA, and their corresponding chromosome copy numbers were measured by ISH, and Ki-67 protein expression was measured by IHC to identify associations with pCR individually and in combination. Figure MET (black) + CEN7 (red) ISH, and PTEN (black) + CEN10 (red) ISH staining, respectively, on selected FFPE sections of biopsy specimens from the neoadjuvant breast cancer cohort. Figure MET (black) and CEN7 (red) by ISH and shows cells with a lesser number of MET signals than CEN7 signals (48 % of cells showed relative MET loss). The tumor had other areas with a greater number of MET signals than CEN7 signals (34 % of cells showed relative MET gain) as well. Figure Gene copy numbers for MET gene copy number relative to chromosome 7 copy number . The parameters representing the percentages of cells with more copies of MET than CEN7 (MET/CEN7 gain) and the percentages of cells with less copies of MET than CEN7 (MET/CEN7 loss) provided larger AUC values , while the parameter based on the sum of the percentage of cells with more copies and less copies of MET than CEN7 (MET/CEN7 gain or loss) provided the largest AUC value (0.791).ISH signals were interpreted using several different parameters to express gene and chromosome copy numbers in order to determine which parameters best associated with pCR. Figure PTEN copies, PTEN gain (AUC = 0.674), the ROC curves of which are plotted in Fig. p-values are included in Table MET/CEN7 gain or loss was further analyzed in combination with Ki-67 expression, the 2 parameters providing the largest AUC values alone. This was done by generating an ROC curve [MET/CEN7 gain or loss was held at its optimal value of 50 % cells while varying the cutoffs for Ki-67 expression .Other parameters showing associations with pCR included Ki-67 expression AUC = 0.726) and the percentage of cells with greater than the normal 2 6 and theOC curve  in whichERBB2/cell, and ERBB2/CEN17). Results are listed in Table MET/CEN7 gain or loss and MET/CEN7 gain or loss combined with Ki-67 expression were significantly associated with age (trend with tumor size), Ki-67 expression was strongly associated with both ERBB2/cell and ERBB2/CEN17, and pCR was significantly associated with only PR expression .Clinical and pathologic characteristics listed in Table In an effort to better identify patients more likely to achieve pCR in patients with ERBB2-positive breast cancer, we have evaluated a series of additional gene and centromere probes as well as Ki-67 expression. In our cohort pCR was achieved in 52 % of patients. As part of the analysis of ISH results we have evaluated different parameters for describing abnormal gene and chromosome copy numbers. This is because there is no single parameter that best describes copy number for all genes and chromosomes for all tumors. For example, gene amplification is often defined as the presence of two or more copies of a gene per copy of the chromosome on which the gene normally resides. This definition of gene amplification was found to have strong clinical relevance with respect to prognosis  in breasMET copy number relative to chromosome 7 copy number. In this series of parameters both gain of MET relative to chromosome 7  and loss of MET relative to chromosome 7  are associated weakly with pCR . However, the sum of cells with relative gain and loss (MET/CEN7 gain or loss) is highly associated with pCR . Furthermore, the average ratio of MET/CEN7 has no association with pCR, either high ratios or low ratios , which is understandable since both MET/CEN7 gain (equating to higher ratio) and MET/CEN7 loss (equating to lower ratio) are associated with pCR and would tend to cancel each other in a ratio calculation. Therefore, proper parameter selection is very important since evaluation of these neoadjuvant specimens using a MET-to-CEN7 ratio, the most common measure of relative copy number, would have erroneously indicated a lack of prognostic value while use of the relative gene-to-chromosome imbalance parameter MET/CEN7 gain or loss provides a high statistical association. Similar to MET and chromosome 7, ISH data for the other genes and corresponding chromosomes were evaluated in terms of the various parameters defined in the Methods section of this paper. Of the other 3 gene probes only PTEN reached statistical significance and this was using PTEN gain in which patient tumors having higher numbers of cells with more than 2 copies of PTEN were associated with pCR . Examining the actual ISH signals per cell and the average PTEN/CEN10 ratios, gain of PTEN was likely a result of chromosome 10 polysomy, with CEN10/cell ranging between 1.5 and 4.2 and PTEN/CEN10 not exceeding a ratio of 1.4 for any one specimen. Several specimens did exhibit PTEN deletion (PTEN/CEN10 < 0.7) but these appeared in both the pCR and non-pCR groups and no association was found for deletion using the parameters of PTEN loss, PTEN/CEN10 ratio, or PTEN/CEN10 loss.The importance of evaluating different parameters can be seen in Table p = 0.0098). The only Ki-67 parameter evaluated was the percentage of cells expressing Ki-67, since this is a standard parameter used by pathologists in the evaluation of breast tissues. Since both MET/CEN7 gain or loss and Ki-67 parameters were highly associated with pCR, the two were analyzed in combination by holding MET/CEN7 gain or loss at its optimal cutoff of 50 % and varying the Ki-67 cutoff over a wide range .Ki-67 expression, as measured by IHC, was also highly associated with pCR  could be informative. One hypothesis is that any alteration of the MET signaling pathway may have some relationship to developing a pCR in the two treatment groups (ACTH and TCH) analyzed in this study. Increased or decreased MET expression potentially resulting from MET gene copy number change might create a fragile condition sensitive to perturbation by therapeutics.MET gene copy number relative to chromosome 7 and Ki-67 expression is strongly associated with pCR in patients with ERBB2-positive tumors treated with neoadjuvant chemotherapy and ERBB2-targeted therapy. Although the use of pCR as a surrogate endpoint for event free (EFS) and overall survival (OS) remains controversial, the ability to predict which patients are most likely to achieve a pCR remains important for individual patient treatment decisions and future clinical trial design. Patients with a sufficiently low likelihood of a pCR who are not candidates for breast conservation at presentation may choose surgery followed by adjuvant chemotherapy and ERBB2-targeted therapy. These data from retrospective studies require validation in a larger, prospective study.Our results show that a predictive score based on


A 50-year-old African American male presented with abdominal pain and significant weight loss. On physical examination, he had parotid and submandibular gland enlargement associated with right eye proptosis. Computed tomography showed a thoracic paravertebral soft tissue mass, enlarged lymph nodes, and ascending aortic aneurysm. Laboratory results were remarkable for elevated total IgG and IgG4 subclass. The submandibular gland pathology revealed chronic sclerosing sialadenitis, with a large subset of inflammatory cells positively staining for IgG4. The histology of the paravertebral mass demonstrated fibrosclerosis with increased lymphocytic infiltrate, associated with increased IgG4 plasma cells. He was diagnosed with immunoglobulin G4-related disease (IgG4-RD). Steroid therapy initially yielded improvement; however, after steroids were stopped, there was disease recurrence. Prednisone was restarted, and the plan was to start him on rituximab. Interestingly, the patient's brother also had IgG4-RD.  IgG4-RD can present as a paravertebral mass which is usually responsive to steroids; however, recurrent and resistant disease can be seen for which steroid-sparing agents such as rituximab should be considered. In addition, to the best of our knowledge, this is the first reported case of IgG4-RD in two family members presenting as a paravertebral mass, highlighting an exciting area for more research in the future. Immunoglobulin G4-related disease (IgG4-RD) is a relatively new multiorgan disease, characterized by an immune-mediated fibroinflammatory process, which can produce irreversible organ damage if untreated –3. The cOver the past few years, there has been increased understating of this condition, and the multiple disorders that were once described as isolated entities are now included under IgG4-RD spectrum , 3. MoreWe present an atypical presentation of IgG4-RD, with not only glandular and ophthalmic involvement but also thoracic paravertebral infiltration. In addition, to the best of our knowledge, this is the first reported case of IgG4-RD in two members of the same family.A 50-year-old African American male with a past medical history of hypertension, gout, sleep apnea, and hypothyroidism was evaluated for abdominal pain, associated with unintentional 20-pound weight loss, nausea, and vomiting. He denied fever, chills, night sweats, headaches, dyspnea, skin rash, arthralgias, oral ulcers, Raynaud's phenomenon, or chest pain.On physical exam, he was found to have bilateral parotid and submandibular gland enlargement and right eye proptosis. The patient disclosed that his brother has retroperitoneal fibrosis secondary to IgG4-RD. The initial abdominal computed tomography (CT) revealed a duodenal mass. Endoscopic retrograde cholangiopancreatography (ERCP) showed a mildly dilated common bile duct, not associated with mass or stricture. A biopsy of the intra- and infra-ampullary area demonstrated active chronic duodenitis without evidence of malignancy.A CT of the thorax showed a confluence of elongated anterior and right paravertebral soft tissue from the midthoracic region to the diaphragmatic level, associated with mildly enlarged thoracic lymph nodes, borderline ascending aortic aneurysm, and scattered small lung nodules .®-TB Gold test was negative.Complete blood count and biochemical profile were remarkable for chronic anemia and hypoalbuminemia, with normal renal and hepatic function. Monoclonal protein evaluation showed elevated IgG level of 5410 mg/dL , and bone marrow biopsy demonstrated plasma cell dyscrasia (plasma cell count of 6%) with no evidence of lymphoproliferative disorder. Additional laboratory results revealed positive antinuclear antibody (ANA), low complement levels, elevated total IgG and IgG4 subclass, and elevated sedimentation rate . The QuaHe underwent excision of a submandibular gland, and the pathology revealed chronic sclerosing sialadenitis, with a large subset of inflammatory cells positively staining for IgG4 .Subsequently, video-assisted thoracoscopy (VATS) was performed. During the procedure, a multilobar paraspinal mass near the diaphragm was noted, with pathology showing dense fibrosclerosis with increased lymphocytic infiltrates. Although staining for IgG and IgG4 was limited by the level of background staining and areas of crush artifact within the tissue, there was an increase of IgG4 plasma cells .A follow-up chest CT revealed a prominent paravertebral mass. In view of this finding, the patient was started on prednisone 40 mg daily. While on prednisone, he significantly improved. A subsequent CT done 5 months later showed a decrease in the paraspinal mass . The serPrednisone was restarted, and rituximab therapy was planned.IgG4-RD is a relatively new systemic fibroinflammatory disease. It is more common in male patients above age 50 , 2. The The diagnosis is based on clinic-pathologic correlation. Clinical manifestations depend on the type and degree of organ involvement. Solid organ involvement commonly presents as an inflammatory pseudotumor , 9. LaboThe gold standard for diagnosis is biopsy, which also can differentiate this entity from other conditions such as lymphoma , 3. The Our patient presented with a paravertebral mass as a manifestation of IgG4-RD. There are only a few case reports that describe this atypical presentation , 10. WatEarly recognition and treatment can prevent permanent organ damage since chronic inflammation leads to a tumefactive mass that can destroy the involved organ , 3. TherIn conclusion, systemic involvement and possible permanent organ damage from IgG4-RD can present insidiously, so all physicians should be aware of this infrequent but important condition since early diagnosis and treatment may alter the prognosis of this multisystem disease. In addition, if there is a family history of IgG4-RD, this should not deter one from considering the diagnosis as our interesting case illustrates.


Immunoglobulin G4 (IgG4)-related disease represents a spectrum of fibro-inflammatory disorders that affects various organ systems, including the central nervous system.Here we present the case of lgG4-related hypertrophic meningitis with exclusively leptomeningeal involvement and spread via perivascular spaces.A 58-year-old male patient presented with complex partial seizures. Initial computed tomography examination showed left frontal sulcal hyperdensity. Subsequent magnetic resonance examination revealed FLAIR hyperintensity in the central sulcus, with post-contrast enhancement in the form of “dotted line.” Physical examination, routine laboratory, and cerebrospinal fluid analyses were unremarkable. Meningeal biopsy confirmed IgG4-related meningitis. After corticosteroid treatment, a complete resolution of imaging findings was observed. Two months later, the patient presented with relapsing neurological symptoms and radiological findings in postcentral, precentral, and temporal sulci, resembling the form of “dotted line” contrast enhancement. In addition, linear intraparenchymal enhancement that followed perivascular spaces was seen in the left parietal lobe. After repeated steroid therapy, all lesions resolved completely.We reported the first case of isolated IgG4-related leptomeningeal involvement with a “dotted line” enhancement and perivascular intraparenchymal spread. Although IgG4-related meningitis represents a rare disease, both clinicians and radiologists should include this condition in the differential diagnosis of unclear leptomeningeal disease. Immunoglobulin G4 (IgG4)-related disease encompasses a spectrum of fibro-inflammatory disorders characterized by IgG4-positive plasma cell infiltration that can affect almost every organ system . The disTo date, several cases of pachymeningitis—but only three cases of leptomeningeal involvement in IgG4-related disease have been described . To the A 58-year-old previously healthy male patient presented with complex partial seizures. Physical examination and routine laboratory tests were unremarkable. Cerebrospinal fluid (CSF) analysis showed a normal cell count, and normal protein and glucose levels. Initial computed tomography (CT) of the brain showed a subtle sulcal hyperdensity in the left frontal lobe Fig. a. Brain IgG4-related disease is a recently recognized spectrum of complex, immune-mediated, sclerosing inflammatory disorders with common pathologic, clinical, and serologic characteristics . The conIgG4-related pachymeningitis generally occurs in men during the fifth and sixth decades. Typical clinical symptoms include headache, seizures or focal deficits, compression or involvement of cranial nerves leading to neuropathies, vessel occlusions, and dural venous sinus occlusions . On CT/MTo date, to the best of our knowledge, no cases of isolated leptomeningeal IgG4-related meningitis with perivascular spread have been described.In our patient, an isolated leptomeningeal involvement was present in the frontal, parietal, and temporal sulci. Leptomeningeal enhancement had an interesting pattern of a “dotted line,” consisting of a small nodular thickening of the leptomeninges. Nodular leptomeningeal enhancement can also be observed in malignant or vasculitic diseases . Also, lSoon after the onset of the leptomeningeal disease, a subtle contrast enhancement was noted in the underlying white matter, along the perivascular spaces, representing inflammatory spread along the perivascular spaces. In addition, T2W/FLAIR signal changes without contrast enhancement were evident in the underlying cortex. These changes probably represented vasogenic edema (no signs of restricted diffusion were present), due to venous congestion and deprived venous outflow. There were no signs of dural involvement or dural sinus thrombosis. Post-contrast T1W images showed enhancement in the narrowed sulci, as in previously described cases of dural thickening –9.There have been only three cases of leptomeningeal involvement in IgG4-related disease reported in the literature , 9, 14. Meningeal biopsy is the gold standard for establishing the diagnosis of IgG4-related disease . DiagnosCSF analysis is performed to exclude other pathologies, such as CNS infections or malignant disease. Usually, the results of CSF analysis reveal normal glucose levels, normal or slightly elevated protein levels, and a variable degree of pleocytosis  . ResultsSerum IgG4 level may or may not be elevated . In our Although currently there is no consensus about the treatment of IgG4-related pachymeningitis, glucocorticoid therapy is the first choice. Recurrent disease after corticosteroid therapy rarely occurs. However, in our case, recurrence was observed after cessation of steroid therapy after a short period of time, similar to the case described by Shapiro et al. . Cases wIn conclusion, this is the first case of isolated IgG4-related leptomeningeal involvement with a “dotted line” enhancement and perivascular intraparenchymal spread. Although a rare disease, both clinicians and radiologists should be aware of this condition as part of the differential diagnosis in patients with leptomeningeal disease. A prompt diagnosis with meningeal biopsy and a specific therapeutic approach might prevent long-term neurological complications.


Psammomys obesus (P. obesus), a rodent model that mimics most of the metabolic features of human diabetes, have not been explored thoroughly. Here, we sought to investigate whether MET might improve insulin sensitivity, glucose homeostasis, lipid profile as well as cellular redox and energy balance in P. obesus maintained on a high energy diet (HED).While metformin (MET) is the most widely prescribed antidiabetic drug worldwide, its beneficial effects in P. obesus gerbils were randomly assigned to receive either a natural diet (ND) consisting of halophytic plants (control group) or a HED (diabetic group) for a period of 24 weeks. MET (50 mg/kg per os) was administered in both animal groups after 12 weeks of feeding, i.e., the time required for the manifestation of insulin resistance in P. obesus fed a HED. Parallel in vitro experiments were conducted on isolated hepatocytes that were shortly incubated (30 min) with MET and energetic substrates .In vivo, MET lowered glycemia, glycosylated haemoglobin, circulating insulin and fatty acid levels in diabetic P. obesus. It also largely reversed HED-induced hepatic lipid alterations. In vitro, MET increased glycolysis but decreased both gluconeogenesis and ketogenesis in the presence of glucogenic precursors and medium-chain fatty acid. Importantly, these changes were associated with an increase in cytosolic and mitochondrial redox states along with a decline in respiration capacity.P. obesus, possibly through a tight control of gluconeogenesis and fatty acid β-oxidation depending upon mitochondrial function. While the latter is increasingly becoming a therapeutic issue in diabetes, the gut microbiota is another promising target that would need to be considered as well.MET prevents the progression of insulin resistance in diabetes-prone  The epidemic nature of type 2 diabetes mellitus (T2DM) has led to its recognition as an urgent priority by the International Diabetes Institute. Indeed, the prevalence of this disease is predicted to reach 5.4% of the worldwide adult population by the year 2025, i.e., 300 million individuals . T2DM isob/ob mice .All the experimental procedures, including Plasma glucose levels were measured by the glucose oxidase test combination . Circulating insulin concentrations were determined by a radioimmunoassay using human insulin as the standard, while ketone bodies, triglycerides and total cholesterol were enzymatically assayed. Plasma lactate concentrations were measured by spectrophotometry and non-esterified fatty acids (NEFA) were determined by microfluorimetry. Glycosylated haemoglobin (HbA1c) was measured by turbidimetry using a Cobas Mira Plus automatic analyser . The hepatic content in triglycerides, esterified cholesterol and free cholesterol was carried out by a sequential quantitative method . GlycogeP. obesus were anesthetized by intraperitoneal injection of sodium pentobarbital (5 mg/100 g), and hepatocytes were then isolated according to the method of Berry and Friend [2PO4, 1.2 mM MgSO4, 24 mM NaHCO3, 2.4 mM CaCl2, 2% BSA, pH 7.4), saturated with O2/CO2 (95:5%). Using a shaking water bath, isolated hepatocytes (10 mg dry cells per mL) were incubated for 30 min at 37°C in closed vials containing 2.5 mL of oxygenated buffer with alanine (20 mM) or lactate + pyruvate (10:1 mM) in the presence of medium-chain fatty acid octanoate (2 mM), with or without MET (1 mM and 10 mM). After 30 min, 350 μL of the cell suspension were deproteinized with HClO4 , then centrifuged at 13,500xg for 5 min. The supernatants were neutralized with KOMO buffer  for subsequent assays of glucose, lactate, pyruvate and ketone bodies  by spectrophotometry [16 h-fasted d Friend , as modid Friend . Liver cotometry . A seconotometry .2/CO2 and immediately transferred into a stirred vessel equipped with a Clark oxygen electrode. The oxygen consumption rate was measured at 37°C and expressed as μmol O2/min/g dry cell.After incubation of hepatocytes with substrates, in the presence or absence of MET as described above, the cell suspension was quickly saturated with Ot test or one-way analysis of variance (ANOVA) as appropriate. The differences were considered statistically significant at p < 0.05.Statistical analysis was performed using SPSS version 12 . Data were expressed as the mean ± SEM, and comparisons between groups were made using student's P. obesus ate over 30.4 ± 2.8 kcal/day (estimated to be around 76 g of food daily or 80% of their body weight) when maintained on an exclusive ND. In such conditions, these rodents do not exhibit hyperglycemia, hyperinsulinemia or plasma lipid impairments. In contrast, the caloric intake was markedly increased in HED fed P. obesus , and their body weights almost doubled when compared to the control group  in MET-treated diabetic P. obesus compared to untreated diabetic gerbils  in this group of animals. The decline was progressive throughout the course of the treatment until reaching a stable end-value (3.88 ± 0.17 mM) near the baseline. Concomitantly, MET reduced the amount of HbA1c. The values in plasma glucose and HbA1c were not BMI-dependent (0.50 < or > 0.60 g/cm2), since they correlated strongly with HOMA-IR and insulinemia in HED fed P. obesus . This lost of sensitivity to insulin in obese animals, as demonstrated by the higher value of HOMA-IR and subsequent hyperinsulinemia, was fully reversed by MET  of in vivo tissue concentrations than the observed blood levels in clinical use. MET used at 1 mM or 10 mM does decrease hepatic glucose ouput in diabetic P. obesus. This dose-dependent effect was observed with different gluconeogenic substrates such as lactate + pyruvate or alanine in the presence of octanoate  prevented the progression of systemic metabolism disturbances and restored insulin sensitivity in diabetic P. obesus, as evidenced by a controlled glycemia and a lowered HOMA-IR. In T2DM and/or obese patients, the primary mechanism for MET action consists of selective inhibition of whole body free fatty acid oxidation while leaving the mitochondrial β-oxidation capability unaffected [P. obesus, a remarkable improvement of hepatic triglyceridemia and cholesterolemia, in association with a decline in plasma NEFA, was observed. The reduced availability of plasma fatty acids together with other coordinated effects on the liver, possibly on the adipose tissue, and potential other effects were tightly related to a MET-driven enhancement of fat metabolism in these diabetic P. obesus, thereby alleviating their advanced complications. Indeed, the inhibition of lipids storage is essential to restore both insulin sensitivity and glycemia in P. obesus fed a regular diet. Importantly, these results were obtained at a dose of MET (50 mg/kg) that was clinically shown to decrease plasma glucose to optimal levels [Previous reports, including ours, have shed light on pronounced physiological disorders in  feeding , 32, 18.ironment , 34. In affected . It was affected . In MET-l levels .Psammomys model, Harmel et al. documented the positive effects of MET on hyperlipidemia associated with insulin resistance and T2DM; these changes began in the small intestine and were dependent on AMPK activity [P. obesus [P. obesus under ND conditions generates short-chain fatty acids  which should in turn activate intestinal gluconeogenesis, providing a source of glucose during starvation or inducing de novo hepatic lipogenesis. Because it was recently established that the bodies of diabetic people have a lower proportion of butyrate-producing bacteria [P. obesus ingesting a HED. Assuming that a modulation of the gut microbiota by MET constitutes a novel component of its antidiabetic action [P. obesus), that MET may alter the gut microbiota diversity of P. obesus in parallel with its effects on host (patho) physiology and that the magnitude of this modulatory effect is probably diet-dependent. This hypothesis is highly interesting and asks for further research.Using this unique activity . Similaractivity . Such obactivity –42. Limi. obesus , 44. Thi. obesus , we suggbacteria , it is lc action , 48, oneP. obesus after MET exposure but lactatemia did not further worsen in diabetic P. obesus given MET. The plasma level of ketone bodies however remained rather high, presumably due to an accumulation of glycogen in the damaged liver of P. obesus. Collectively, our in vivo findings suggest the relevance of MET action on the crossroads between lipid and glucose metabolism. This could be partly mediated by intestinal and liver mechanisms without ruling out the implication of circulating redox state since it was found that altered plasma lactate/pyruvate ratios can regulate liver metabolism in mice [Regarding other issues for MET action, we were struck by the consistent observation of increased plasma lactate amounts in drug-treated animals and patients , 50. It  in mice .in vitro results, achieved by contrast with MET concentrations (millimolar) higher than those applied in human therapy, which excluded any role of gut microbiota at the cellular level, we assured that MET ameliorated hyperglycemia in P. obesus through inhibition of gluconeogenesis from various energizing substrates. Lactate + pyruvate supplied reducing equivalents  from glycolysis to the mitochondrial respiratory chain and, without modifying regulation of the metabolic routes; the cytosolic redox potential was imposed using a concentration ratio for these substrates of 10:1 [in vitro study was the link between ketone body production and fatty acid ß-oxidation in MET-treated diabetic P. obesus. If the decrease in ketone bodies could be accounted for by a reduction in octanoate oxidation, at least partly (ketogenesis reflecting in fact the flux through fatty acid ß-oxidation), the results were rather in favour of an enhanced ß-oxidation. Owing to the inhibition of gluconeogenesis by MET, the amount of oxaloacetate available for the reaction with acetyl-CoA increases, and acetyl-CoA is then preferentially channelled from entering the Krebs cycle at the expense of the pathway producing acetoacetate and β-hydroxybutyrate. This kind of process can operate because MET acutely inhibited oxygen consumption rates in hepatocytes from diabetic P. obesus, thereby decreasing ATP content and increasing redox potential. To re-oxide the reducing equivalents in the case of diminished glucose synthesis, MET not only stimulated glycolysis (by increasing lactate) but also enhanced fatty acid β-oxidation concomitantly with an inhibition of ketogenesis. All these statements were consistent with the fact that MET facilitated the removal of plasma NEFA and improved hepatic lipid metabolism in diabetic P. obesus.Based on  of 10:1 . Alanine of 10:1 . Octanoa of 10:1 . In our  of 10:1  or an en of 10:1 . AnotherP. obesus [P. obesus, this drug induced mixed effects in the latter. Indeed, gluconeogenesis was barely affected and glycolysis was increased but both pathways were significantly altered in Wistar rats [P. obesus [MET was revealed to inhibit mitochondrial glycerol-3-phosphate dehydrogenase, raising the cytosolic NADH/NAD+ ratio and impairing utilization of redox-dependent substrates for gluconeogenesis, but surprisingly the mitochondrial NADH pool decreased . Here as. obesus , the curtar rats . This ma. obesus , thus pe. obesus , 63.P. obesus. Meanwhile, the caloric intake was substantially lessened by MET. This drug could thus indirectly influence the central nervous system through a modulation of hypothalamic feeding circuits [P. obesus rats [via the gut as newly proposed in human beings, at least when administrated orally [P. obesus under MET treatment could be linked to this phenomenon but direct evidence for this was not available.Although some studies found that MET reduced body weight gain in patients suffering from diabetes , we did circuits . Indeed,circuits  and T2DMcircuits . In thatsus rats . These ld orally . An explP. obesus did not fulfill the criteria for metabolic syndrome, by means of adaptation to a hypocaloric food (ND), transferring them to a HED for 24 weeks led to profound metabolic disorders which were acutely corrected by MET. Our in vivo and in vitro findings confirmed its effectiveness to ameliorate ketoacidosis and lipid metabolism in diabetic P. obesus; these events likely relied on hepatocellular redox status and mitochondrial activity (see If healthy P. obesus and human pathology should help explain, at least partially, the higher risk of developing morbid obesity for people with unhealthy dietary habits. In addition, part of the MET taken orally by P. obesus might directly interact with the microbiota of the gastrointestinal tract before entering the circulation and peripheral tissues to protect host cells against severe damages. This open question linked to the dependency and interaction of MET effects with microbiome in metabolic diseases needs to be further elucidated.This study likewise agreed with the successful use of MET for patients intolerant to glucose or with moderate hyperglycemia. Such data are of utmost importance for establishing innovative therapeutic strategies, and any comparison between the natural history of diabetes in


Chronic scapholunate ligament (SL) injuries are difficult to treat and can lead to wrist dysfunction. Whilst several tendon reconstruction techniques have been employed in the management of SL instability, SL gap reappearance after surgery has been reported. Using a finite element model and cadaveric study data, we investigated the performance of the Corella, scapholunate axis (SLAM) and modified Brunelli tenodesis (MBT) techniques. Scapholunate dorsal and volar gap and angle were obtained following virtual surgery undertaken using each of the three reconstruction methods with the wrist positioned in flexion, extension, ulnar deviation and radial deviation, in addition to the ulnar-deviated clenched fist and neutral positions. From the study, it was found that, following simulated scapholunate interosseous ligament rupture, the Corella technique was better able to restore the SL gap and angle close to the intact ligament for all wrist positions investigated, followed by SLAM and MBT. The results suggest that for the tendon reconstruction techniques, the use of multiple junction points between scaphoid and lunate may be of benefit. Scapholunate interosseous ligament (SLIL) injury is a relatively common , 2 wristIn flexion-extension of the wrist, the lunate rotates over the radius in the dorsal direction during flexion and in the volar direction during extension ; therefoMore recently, techniques including Corella  and scapIn this study, the finite element method together with in vitro cadaveric tests were used to investigate the performance of the modified Brunelli tenodesis (MBT), Corella and SLAM reconstruction methods in regard to their ability to restore wrist stability  followinOnce the models had been validated, an investigation of the performance of the three reconstruction methods , the intact (ligament) and the SLIL sectioning cases was undertaken with the wrist positioned at 20° flexion and 20° extension, at 15° ulnar deviation and 15° radial deviation. The predicted values of SL angle and SL gap at both dorsal and volar sides obtained from these models were used for comparison purposes between the reconstruction techniques.The finite element method is widely employed for undertaking analyses in biomechanics offering a number of well-documented advantages compared to cadaveric studies including repeatability of analyses, ease of study parameter modification and lack of associated ethical issues. A particular advantage in utilising the finite element method in our study was that it facilitated calculation and comparison of both dorsal and volar angles for all wrist positions analysed, which is not currently feasible with the radiograph-based techniques currently employed for cadaveric/clinical studies.3-D finite element (FE) models of the right wrist from a 63-year-old female were created using 133 slices of computer tomography (CT) scan data. The slices were of 0.7 mm thickness and the transverse resolution was 512 × 512; the pixel size was 0.289 × 0.289 mm. 3-D image data processing software was used to create the surfaces of the geometry from the CT scan data. In addition, to optimise the element size of the mesh, data resampling using a pixel spacing of 0.4 × 0.4 × 0.4 mm was performed.Reconstruction of each of the 15 cortical bone surfaces considered in the 3-D FE models was performed using a thresholding method. Masks created in the image data processing software to describe the cortical bone were exported as 3-D geometries and imported into Abaqus 6.14 . The metacarpal bones were fixed and only a cortical shell was included as these geometries do not come into contact with the scaphoid and lunate bones, the main bones of interest in this study. For the remaining bones, once the cortical layer was defined, the internal volume was filled virtually and assigned trabecular bone material properties. The cortical layer thickness was determined from the CT scan and varied for each bone; the average thickness for all the bones was 2.35 mm. Metacarpal bones have an average thickness of 2.4 mm, radius and ulna 3.0 mm and the carpal bones 1.65 mm .The Abaqus 6.14 software was used to create the solid mesh from the surfaces of the cortical bone geometry imported previously. Linear tetrahedral elements (C3D4) were used to mesh the bone geometry. Linear tetrahedral elements were considered adequate for the analysis in this case as the study was primarily concerned with predicting the relative position of the bones as a result of various movements rather than obtaining accurate stress estimates. The same software was used to assemble the meshed bone geometry in order to create the wrist model. Wedge elements (C3D6) were used to simulate the cartilage for the articulation between bones. The thickness of the cartilage was obtained by taking half of the distance between two articulating bones , 13. TwoThe FE models were created based on the intact (ligament) wrist model. The models simulate three reconstruction techniques; the scapholunate axis method (SLAM), Corella and MBT, with the hand in the neutral position, in the ulnar-deviated clenched fist position, at 20° flexion, 20° extension, 15° radial deviation and 15° ulnar deviation. In addition, for each wrist position, models were created to simulate the wrist joint following SLIL sectioning, where the connection between the scaphoid and lunate bone is severed  with the internal surfaces of the scaphoid/lunate. The cylindrical hole drilled through the bone was included in the model in order to give a more accurate representation of the surgical procedures. For the intact model, in all positions, the scaphoid and lunate were joined with SLIL Fig. Hand in the neutral position: SLIL ligament intactUlnar-deviated clenched fist position: SLIL ligament intactUlnar-deviated clenched fist position: SLIL sectionedUlnar-deviated clenched fist position: wrist following MBT tendon graft surgeryUlnar-deviated clenched fist position: wrist following SLAM tendon graft surgeryUlnar-deviated clenched fist position: wrist following Corella tendon graft surgeryThe finite element wrist models were validated by comparing model predictions with data from a cadaveric study. Six scenarios were considered for the validation:Fifteen cadaveric hands and wrists from specimens with a mean age of 75 years, range 54 to 94, were used for this study. The specimens were sectioned at the mid-forearm. To measure SL  gap and angle of the specimens , 23, theTo be able to reproduce the motion considered in this study, six tendons of the cadaver wrists were exposed: flexor carpi ulnaris (FCU), flexor digitorum superficialis (FDS), flexor digitorum profundus (FDP), flexor pollicis longus tendon (FPL), extensor carpi ulnaris (ECU) and extensor digitorum communis (EDC). Five tendon groups  were created using FiberWire locking stitches. The ulnar-deviated clenched fist position was created by first cementing the cadaveric hands in a cylindrical plastic container which was then fixed on the edge of a table utilising a clamp. Loads of 15 and 20 N were hung separately from FDP/FPL, FCU, FDS, ECU and EDS tendons Fig. .Fig. 3TeThe SLIL of all the cadaveric hands was sectioned using a scalpel blade in order to reproduce the scapholunate instability. For the position of the ulnar-deviated clenched fist, the angle and SL  gap were determined before and after sectioning through the PA and lateral plain radiograph stress views , 23. Speα)  Fig. . These vInitial model validation consisted of comparing joint contact area and contact pressure predictions from our intact (ligament) wrist model with those determined experimentally in the cadaveric study undertaken by Tencer et al. . The speOur model predicted a contact area/total area ratio of 0.182 compared to 0.206 ± 0.0495 in the cadaveric study and a scaphoid/lunate contact area ratio of 3.02 compared to 3.72. The peak joint contact pressure predicted by the model, 4.52 MPa, was in the range determined in the cadaveric study, 2–5.6 MPa.A mesh sensitivity analysis was undertaken to investigate model convergence. Mesh density was increased in the loaded intact ligament model until SL gap and angle changed by less than 1%. This density was then employed for subsequent analyses.The results of the extended model validation exercise are shown in Fig. A comparison of experimentally determined and FE model-predicted SL angle is presented in Fig. It is interesting to note that following SLIL sectioning, the SL gap increased significantly under loading compared to the intact loaded ligament case, by 1.2 mm (50%) in the experimental study and by 1.8 mm (90%) in our numerical model. In contrast, SLIL sectioning had less effect on SL angle, resulting in a mean reduction of just 3.2° (6%) in the cadaveric case and an increase of 1.4° (2.8%) in the FE model compared to the intact loaded ligament case. Tendon reconstruction reduced  SL gap back to the original intact loaded ligament values or below in the cadaver experiments and to within 10% for our FE model cases. In addition, tendon reconstruction resulted in an SL angle less than the corresponding intact loaded ligament value in both the cadaver and FE model studies.Figure Following application of the three reconstruction techniques, SL gap was restored closer to that of the intact ligament, to within 10.5% for Corella and SLAM and 26% for MBT. In terms of dorsal SL gap for the clenched fist posture  or less of the intact. It is also worth noting that in extension, the MBT method resulted in the same size of volar SL gap as that obtained for the SLIL-sectioning case, 3.2 mm or 28% greater than that of the intact. SL angles were greater than the intact for the three reconstruction techniques and the SLIL-sectioning case in flexion whereas the converse was true in extension, where the SL angle was always lower than for the intact ligament.Figures Upon inspection of Figs. Overall, of the tendon reconstruction methods simulated, the Corella method resulted in a dorsal and volar SL gap and SL angle closer to that of the intact ligament case for radial and ulnar deviation positions of the wrist.Application of the Corella technique restored volar SL gap to that of the intact ligament (2.5 mm), dorsal SL gap to within 5% and SL angle to within 0.5% of the intact ligament values.Of the SLAM and MBT methods, SLAM was better able to restore dorsal and volar SL gap, restoring SL gap to within 21% of the corresponding intact ligament values for radial and ulnar deviation. However, the MBT tendon reconstruction technique was able to restore SL angle closer to that of the intact, within 1%, compared to 3.5% for SLAM.Dorsal SL gap varied least amongst the reconstruction techniques in ulnar deviation, with all three techniques able to restore dorsal SL gap to within 0.1 mm (5%) or less of the intact. Volar SL gap varied greatest amongst the reconstruction techniques in ulnar deviation; whilst the Corella technique was able to restore the volar SL gap to that of the intact ligament, the SLAM and MBT techniques produced a volar SL angle that was 21% and 32% greater than the intact case, respectively.SL angle was equal to or lower than the intact case for the three reconstruction techniques, whereas the angle was greater for SLIL sectioning.A variety of treatments currently exist for treating chronic SL instability. Ligamentous reconstruction techniques including capsulodesis, bone-ligament-bone and tenodesis are an option where patients present with non-repairable SLIL injury but a reducible SL dissociation . Until rUsing a finite element model and cadaveric study data, we investigated the performance of the Corella, SLAM and MBT techniques.SL gap and angle predictions from our model were in good agreement with those from the cadaveric study for the six scenarios considered, including the three ligament reconstruction techniques MBT, SLAM and Corella. SL gap predictions were all within 0.4 mm and SL angles within 7.7° of the corresponding experimental mean data values. The cadaveric data and predictions from our model showed that SLIL sectioning had a much greater effect on SL gap compared to angle, with experimentally obtained mean SL gap increasing by 50% or 1.2 mm compared to the intact ligament case for the ulnar-deviated clench fist position. In this case, SL gap values met the criteria for SL dissociation . In contFor the ulnar-deviated clenched fist posture investigated, the tendon reconstruction techniques reduced dorsal SL gap to within 10% of the values obtained for the original intact loaded ligament and volar SL gap to within 26% in all cases whilst maintaining SL angle in the normal range, demonstrating the techniques’ abilities to restore dorsal SL gap following non-repairable SLIL injury. Of the three tendon reconstruction techniques, Corella was more effective in restoring SL gap, restoring dorsal gap back to the original intact loaded value and volar gap to within 10.5%.In flexion-extension, the lunate rotates over the radius “in the dorsal direction” during flexion and “in the volar direction” during extension ; therefoThe predictions from our FE models indicated that for radial and ulnar deviation, all three reconstruction techniques simulated were able to restore dorsal SL gap to within 0.3 mm and SL angle to within 1.8° of the intact ligament following SLIL sectioning. Again, this is not unexpected as all techniques involve dorsal SLIL portion reconstruction. However, more variation was found in the ability of the techniques to restore dorsal SL gap. The Corella technique, which involves reconstruction of the volar portion of the SLIL, restored volar SL gap back to that of the intact ligament for both radial and ulnar deviation. SLAM, which involves a multiplanar scaphoid-lunate tether, was able to restore volar SL gap to within 4.5% of the intact ligament in radial deviation and 21% in ulnar deviation, whereas the corresponding values for MBT, which reconstructs only the dorsal portion of the SLIL, were 9% and 32%, respectively.The relative significance of the various portions of the SLIL is still undecided, with conflicting data available in the literature . HoweverLimitations and assumptions apply to our study which are typical of complex numerical analyses in the field of biomechanics. A number of assumptions and simplifications were inevitably required including geometrical representations and material properties. In terms of soft tissue representations, a hyper-elastic material model was employed for cartilage which is considered to provide a more accurate representation of behaviour , 13.The majority of the ligaments included in the model were represented using spring elements. Whilst the use of one-dimensional representations of ligament geometries is commonplace in biomechanical joint models and has been shown to be valuable particularly for investigating kinematics where external loading is present, a number of limitations have been identified, including the inability to accurately capture non-uniform 3-D stress and strain, non-uniform deformations and joint orientation effects . Three-dWe validated our finite element wrist model by comparing predicted SL gap and SL angle with data obtained from an in vitro cadaveric study conducted on intact, SLIL-sectioning specimens and cadaveric wrists following simulation of the three reconstruction techniques with the hand in the neutral position and under ulnar-deviated clenched fist posture. The good agreement between predicted and experimentally obtained SL gap and angle data suggests that our model is able to model and represent behaviour to a good degree of accuracy and the assumptions used in the model do not introduce significant error.We used CT scans from the wrist of a single volunteer for creating our FE models; therefore, caution should be taken from drawing extensive conclusions from the results. That said, model predictions compared well with mean results from simulation of the three reconstruction techniques undertaken on 15 cadaveric wrists; therefore, our wrist model appears to be a good representation and a certain degree of confidence can be placed in the results from it.Although SLIL sectioning only partly replicates scapholunate instability, it is recognised that the SLIL is the primary stabiliser of the joint . To bettFixation of tendon reconstructions to bone is treated similarly in every reconstructive option we modelled , which mSL gap and angle measurement of the cadaveric wrist specimens was based on posteroanterior (SL) and lateral (angle) plain radiographs and therefore any rotational error in X-ray positioning could potentially affect accuracy.


MET amplification (NCT01657214). SAR125844 was administered by intravenous infusion (260–570 mg/m2) on days 1, 8, 15, and 22 of each 28-day cycle. Objectives were to determine the maximum tolerated dose (MTD) and to evaluate SAR125844 safety and pharmacokinetic profile. Antitumor activity was also assessed. Of 38 patients enrolled (median age 64.0 years), 22 had gastric cancer, including 14 with MET amplification. In the dose-escalation cohort , the MTD was not reached, and the recommended dose was established at 570 mg/m2. Most frequent treatment-emergent adverse events (AEs) were nausea (36.8%), vomiting (34.2%), decreased appetite (28.9%), and fatigue or asthenia, constipation, and abdominal pains (each 21.1%); none appeared to be dose-dependent. Grade ≥ 3 AEs were observed in 39.5% of patients and considered drug-related in 7.9%. SAR125844 exposure increased slightly more than expected by dose proportionality; dose had no significant effect on clearance. No objective responses were observed in the dose-escalation cohort, with seven patients  having stable disease. Modest antitumor activity was observed at 570 mg/m2 in the dose-expansion cohort, comprising patients with MET-amplified tumors (N = 19). Two gastric cancer patients had partial responses, seven patients had stable disease (six gastric cancer and one kidney cancer), and 10 patients had progressive disease. Single-agent SAR125844 administered up to 570 mg/m2 has acceptable tolerability and modest antitumor activity in patients with MET-amplified gastric cancer.SAR125844 is a potent and selective inhibitor of the c-Met kinase receptor. This was an open-label, phase I, multicenter, dose-escalation, and dose-expansion trial of SAR125844 in Asian patients with solid tumors, a subgroup of whom had gastric cancer and  MET proto-oncogene, and its ligand, the hepatocyte growth factor (HGF), trigger a broad spectrum of biologic processes involved in tumorigenesis , , MET genMET-amplified human gastric tumors, intravenous treatment with single-agent SAR resulted in c-Met kinase inhibition and dose-dependent tumor regression. SAR also had an acceptable toxicity profile ) increased slightly more than dose proportionally over the 260- to 570-mg/m2 dose range: a 2.19-fold increase in dose resulted in a 3.00- and a 2.69-fold increase in AUC0–168, on days 1 and 22, respectively. This finding is reinforced by the results of the statistical analysis, which showed no significant effect of dose on total body clearance  or CL at steady state . Pharmacokinetic parameters were similar on days 1 and 22, and no accumulation was observed between days 1 and 22. SAR exhibited a large volume of distribution at steady state (approximately 253 L at the RD).Profiles of the mean blood concentration of SAR over time on days 1 and 22 of cycle 1 are presented in Figure 2 to 1.7 months at 440 mg/m2.No objective responses were observed in the dose-escalation cohort (unselected population). Although 7 patients  had stable disease after 1 month of treatment, all seven stopped treatment within the subsequent 2 months because of disease progression; 11 patients had early disease progression leading to treatment discontinuation in cycle 1. Best overall response and median duration of treatment by dose cohort are shown in MET-amplified tumors (dose-expansion cohort), two patients (10.5%), both with gastric cancer, had a confirmed partial response, and one patient  had an unconfirmed partial response. These three responders had high levels of MET amplification (≥ 97% amplified cells). However, given the small sample number, no correlation could be identified between the level of MET amplification and best change from baseline in tumor measurement  in gastric cancer patients with MET amplification. Notably, this preliminary efficacy was observed in tumors that had a high level (≥ 97%) of MET amplification.This study aimed to establish the RD of a weekly administration of SAR in Asian patients with solid tumors. No DLTs were observed in the dose-escalation phase, and 570 mg/mulations . SAR hadulations –12 and mMET gene amplification. Trials of tivantinib, a c-Met inhibitor, and foretinib, a c-Met/vascular endothelial growth factor receptor 2 inhibitor, in unselected gastric cancer patients have shown no objective responses [MET-amplified (MET:CEP7 >5) gastroesophageal cancers [MET-amplified gastroesophageal junction, gastric, or esophageal tumors [in situ hybridization (FISH) to identify whether a tumor was MET amplified or not. However, the cut-off for defining MET gene amplification differed. The studies of tivantinib and crizotinib considered tumors with a ratio of MET:CEP7 ≥ 2.2 as MET amplified [MET:CEP7 ≥ 2 to define MET amplification [MET amplification is not provided [Studies of other c-Met inhibitors in gastric cancer have shown a variety of efficacy outcomes, and differences are probably related to population heterogeneity and the definition of esponses , 14. How cancers . Treatmel tumors . All stumplified , 15, wheprovided .Combination therapy of c-Met-targeted antibodies and first-line chemotherapeutics in gastric cancer has shown no clear clinical benefit. For example, addition of onartuzumab (an anti-c-Met antibody) to chemotherapy provided no survival advantage, regardless of c-Met expression , and rilMET amplification [MET amplification in gastric cancer is a barrier to the inclusion of large-enough numbers of patients in trials. Also, stringent methods for assessing amplification must be used. A recent study using next-generation sequencing revealed that amplification of receptor tyrosine kinases does not correlate with protein expression in gastric cancer, and a comprehensive analysis using both next-generation sequencing and immunohistochemistry may be necessary to adequately select patients [MET amplification, will likely use a high threshold for the definition of amplification [MET amplification in distinct tumor lesions within a single gastric cancer patient has been reported, and this heterogeneity may be a key driver of resistance to c-Met inhibitors [Preclinical studies provide a wealth of data demonstrating antitumor activity of small-molecule c-Met kinase inhibitors –22. In tfication , 16. Howpatients . Additiofication , and wilhibitors .MET gene amplification (dose-expansion phase), ECOG PS of 0–2, and adequate hematologic, hepatic, and renal function. Exclusion criteria included previous treatment with any c-Met inhibitor, unresolved grade > 1 toxicity  related to any prior anticancer therapy, a washout period of < 3 weeks since previous antitumor therapy or investigational treatment (or < 6 weeks if the previous treatment was nitrosourea or mitomycin C), or known hypersensitivity/experienced any AE related to the study-drug excipient Captisol® . All patients provided written informed consent.Asian patients were recruited from multiple centers. Key eligibility criteria were age ≥ 20 years, a solid tumor for which no standard therapy was available  plus measurable disease and MET-amplified tumors, including gastric cancer).This was an open-label, phase I, multicenter study consisting of a dose-escalation phase (to determine the MTD of SAR) and a dose-expansion phase  was controlled . Dose escalation did not proceed until at least three patients had been treated at the lower dose level. The model was run based on DLTs occurring during the first cycle of treatment.2, and dose increments of 30% were made, to a maximum of 570 mg/m2. Treatment continued until disease progression, unacceptable toxicity, or consent withdrawal. Patients were screened within 21 days before the first administration and followed for 30 days after the last administration.SAR was administered by weekly intravenous infusion on days 1, 8, 15, and 22 of each 28–day cycle. The starting dose was 260 mg/mThe study was approved by the institutional review board or independent ethics committee of each participating center, and was conducted in accordance with the Declaration of Helsinki and the International Council for Harmonisation Guideline for Good Clinical Practice.The MTD was defined as the dose having the highest probability of generating 20‒35% of study drug-related DLTs, and was primarily determined based on DLTs observed in cycle 1. A DLT was defined as grade 4 neutropenia for ≥ 7 consecutive days, febrile neutropenia, grade 4 thrombocytopenia or grade 3 thrombocytopenia with bleeding requiring a transfusion, any grade ≥ 3 nonhematologic event not easily managed or corrected by medical intervention, or SAR-related toxicity resulting in two or more missed doses.In the absence of DLTs at the highest administered dose, relevant information from the first-in-human study  was consmax), AUC from 0 to infinity, AUC0–168, CL, CL after repeated administrations (CLss), volume of distribution at steady state (Vss), and the half-life associated with the terminal slope (t1/2z).Blood samples for pharmacokinetic analysis were acquired using dried blood-spot technology on days 1 and 22 of cycle 1 at several time points before, during, and after infusion (up to 168 hours after administration). Drug concentrations were determined by a validated method of liquid chromatography and tandem mass spectrometry (limit of quantification 2 ng/mL). Pharmacokinetic parameters were calculated using a noncompartmental analysis and included the maximum observed blood concentration  were to be treated at the MTD (expansion cohort). Descriptive statistics were used to summarize patient characteristics, safety, exposure, and antitumor activity.


Inappropriate use of antibiotics is contributing to the increasing rates of antimicrobial resistance. Several Danish guidelines on antibiotic prescribing for acute respiratory tract infections in general practice have been issued to promote rational prescribing of antibiotics, however it is unclear if these recommendations are followed. We aimed to characterise the pattern of antibiotic prescriptions for patients diagnosed with acute respiratory tract infections, by means of electronic prescriptions, labeled with clinical indications, from Danish general practice. Acute respiratory tract infections accounted for 456,532 antibiotic prescriptions issued between July 2012 and June 2013. Pneumonia was the most common indication with 178,354 prescriptions (39%), followed by acute tonsillitis (21%) and acute otitis media (19%). In total, penicillin V accounted for 58% of all prescriptions, followed by macrolides (18%) and amoxicillin (15%). The use of second-line agents increased with age for all indications, and comprised more than 40% of the prescriptions in patients aged >75 years. Women were more often prescribed antibiotics regardless of clinical indication. This is the first Danish study to characterise antibiotic prescription patterns for acute respiratory tract infections by data linkage of clinical indications. The findings confirm that penicillin V is the most commonly prescribed antibiotic agent for treatment of patients with an acute respiratory tract infection in Danish general practice. However, second-line agents like macrolides and amoxicillin with or without clavulanic acid are overused. Strategies to improve the quality of antibiotic prescribing especially for pneumonia, acute otitis media and acute rhinosinusitis are warranted. Better adherence to guidelines for prescribing antibiotics for different respiratory tract infections are warranted in Danish general practice. The over-use of antibiotics, particularly so-called ‘second-line’ agents such as amoxicillin, increases resistance and may lead to a potentially catastrophic scenario where antibiotics are no longer effective. Exactly how widespread the over-use of antibiotics is for different infections, however, is not clear. Rune Aabenhus at the University of Copenhagen and co-workers analyzed primary care data regarding antibiotic prescriptions for acute respiratory tract infections including pneumonia and ear infections in Denmark. They found that penicillin V—the current recommended first-line drug in Scandinavian countries—accounted for 58 per cent of prescriptions, a figure which should be improved. Amoxicillin and macrolides were over-prescribed, particularly in elderly patients. The team also call for further analysis of prescriptions given by out-of-hours clinics. Strategies, such as guidelines on rational antibiotic use, as well as educational and decision support systems are applied to improve antibiotic prescribing. However, the implementation of these strategies into daily clinical routine can be difficult and daily practice is far from optimal8 in turn augmenting the risk of inappropriate prescribing both in regard to quantity  and quality .9Acute respiratory tract infections are common reasons for consulting in general practice and assumed responsible for more than 60% of the antibiotic use in this setting.10 National Guidelines11 on antibiotic prescribing for acute respiratory tract infections recommend narrow-spectrum penicillin (penicillin V) as first-line agent for acute respiratory infections, except acute exacerbations of chronic obstructive pulmonary disease (AECOPD) where the recommendation was amoxicillin with clavulanic acid (co-amoxicillin).As antibiotic resistance rates are low in Denmark12In Denmark, previous attempts to assess current prescribing practice for specific infections have been based on small subsets of general practitioners that volunteered to participate for a limited time.13 Patterns of antibiotic prescriptions, particularly when linked to clinical indications and patient demographics, can be helpful in determining appropriateness of the issued prescribing: lately results from the UK and the Netherlands have successfully applied data linkage to characterise prescribing of flucloxacillin and analyze trends in prescribing for sore throat, colds, and acute cough.16Surveillance of antibiotic prescribing and consumption is a central element in containing the increasing antimicrobial resistance.To promote rational antibiotic use in Denmark, and study the link between clinical indications and the associated antibiotic prescribing, an electronic system with mandatory data entry of clinical indications for antibiotic prescribing was introduced in 2011.We aimed to characterise the pattern of antibiotic prescriptions for acute respiratory tract infections, by means of clinical indications from electronic prescriptions, in regard to national guidelines on antibiotic prescribing in Danish general practice.N = 96.549; 21%), acute otitis media , acute rhinosinusitis , AECOPD , and acute bronchitis   corresponding to 424 treatments per 1000 inhabitants were issued in the 1-year study period. A clinical indication was assigned in 1.6 million prescriptions, however about 25%  of the prescriptions did not include a specific clinical indication, but only stated e.g. “against infection”. Acute respiratory tract infections accounted for 456,532 prescriptions, of which pneumonia accounted for 178,354 (39%), followed by tonsillitis  1.32–1.34). Notably, women were prescribed antibiotics twice as often as men  in acute rhinosinusitis.Marked variations were found in relation to age Fig. . PrescriIn total, penicillin V accounted for 57.7% of all prescriptions; macrolides second 18%) and amoxicillin third (15%). Quinolone and tetracycline use were low. Antibiotic use patterns differed between the individual clinical indications , followed by acute tonsillitis (21%), AOM (19%), and acute rhinosinusitis (18%). The frequency of the clinical indications showed pronounced associations with age. We found that penicillin V was the most commonly used antibiotic agent (58%), macrolides second (18%), and amoxicillin third (15%).17 increased, especially in the older age group. Further efforts are needed to promote rational antibiotic use in patients with acute respiratory tract infection, particularly for AOM, acute rhinosinusitis, and pneumonia.The relative low use of penicillin V is not in line with guidelines recommendations on acute respiratory tract infections (except AECOPD) indicating that current prescribing could still be improved in order to better adhere to Danish guidelines on antibiotic use. Importantly, we found that the use of second-line agents including critical macrolides that belong to the WHO-defined group of antibiotics of the highest priority18 No clear evidence has pointed to a specific antibiotic agent as most effective in empirical antibiotic therapy.19 As most serious acute respiratory infections are related to pneumococci, penicillin V may (in the absence of high resistance rates) be regarded a rational choice due to its high activity against gram positive cocci  20 and few side effects.Few non-Scandinavian countries recommend penicillin V as first-line agent and the European Society for Clinical Microbiology and Infectious Disease recommend amoxicillin or a tetracycline in lower respiratory tract infections.21 and this is most probable related to the superior taste of amoxicillin compared to the first-line agent penicillin V. Accordingly, it may be reasonable to prescribe amoxicillin to secure compliance with treatment,22 also as recent studies have suggested that resistance selection by amoxicillin is mostly short-lived (less than 1 month).23 However the long-term effects and increased risk of resistance24 calls for caution, and antibiotic prescribing should be targeted to patients with a high chance of benefit.25In young children, who for the most part will use syrup or other liquid formulations of antibiotics, the high use of amoxicillin is also reported in studies from Norway26 Furthermore, less than 6% of the antibiotic prescriptions for acute tonsillitis were administered in children under 3 years, a group having a low a priori risk of strep throat.27 In Dutch children, the use of first-line treatment with penicillin V in acute tonsillitis was 60%.8 Most Danish general practitioners (GPs) use point-of-care tests to swab for the presence of Streptococcus group A antigen as part of the clinical management of sore throat. Identifying a causable pathogen may lead to choosing a relevant narrow-spectrum agent such as penicillin V.Antibiotic use for patients diagnosed with acute tonsillitis was predominantly penicillin V (86%),which may be perceived as in line with national recommendations.6 Still two-thirds of all prescriptions are given in children older than 2 years of age and half in patients aged more than 5 years. This finding indicates a possible overuse of antibiotics in patients diagnosed with AOM, which is a common reason for antibiotic use in children.16Today, AOM is mostly perceived as a self-limiting disease in children older than 2 years of age and the modest benefit of antibiotic treatment has been established.28 In adults, the antibiotic use in acute sinusitis has previously been assessed as inappropriate as 60% have had less than 5 days of symptoms prior to presentation and only 30% presented with fever.29In contrast, age-stratified antibiotic prescribing in patients diagnosed with acute rhinosinusitis appeared meaningful as only 3% of prescriptions were issued in children under 15 years of age. Children do not have fully developed sinuses until adolescence. However, it is important not to miss this diagnosis due to the risk of orbital or endo-cranial complications, especially in young children under 6 years of age.www.statistikbanken.dk). It has previously been estimated that the number of patients seen in general practice was up to five times greater, which mounts to roughly the number of clinical indications “pneumonia” the same year (178.354). This number may however be affected by the limited available indications in the electronic prescription system  or due to diagnostic drift, i.e., that the doctor tends to justify a prescription by labeling it”pneumonia”.30 Secondly, GPs may have entered a free text indication or selected an unspecific term “infection”, as such not permitting a detailed analysis of the prescribing. Lastly, some cases of mild pneumonia may not have been prescribed antibiotics or perhaps the patient never filled the prescription (primary non-compliance in Denmark is 6.5% for antibiotic scripts).31 This may indeed be the case as results from the European project Happy Audit32 found 17% of acute respiratory tract infections to be acute bronchitis with an associated antibiotic prescribing rate of 35%. The striking differences to the national figures based on the clinical indications we present in this paper is problematic, as the current system of electronic prescriptions may not capture a sufficiently detailed picture of antibiotic prescribing.The clinical indication “pneumonia” made up 39% of all antibiotic prescriptions for acute respiratory tract infections. In 2012, there were 30,000 hospital admissions with pneumonia and 1555 patients died . These findings are in line with the Danish recommendations stating that these agents should only be prescribed following microbiological identification and known susceptibility to treatment.34 Moxifloxacin was the main quinolone prescribed for AECOPD and acute rhinosinusitis, while ciprofloxacin was issued for patients diagnosed with pneumonia or AOM.Prescribing of other critical antibiotics of the highest priority,The broader the antibiotic spectrum an agent has, the larger the effect on the intestinal flora. Penicillin V is considered a narrow-spectrum antibiotic, but most second-line agents have a broader spectrum of antibacterial activity. Avoidance of inappropriate use of second-line agents is a requirement to limit harm to the patients’ normal flora and minimize risk of developing antibiotic resistance in common bacteria.The electronic data collection ensured complete and valid routine data, from GPs not aware of the investigation, making the results generalizable to other settings.15 Also, the number of treatments per age group provided should be interpreted with caution as missing and unspecific indications are not included. However, the total antibiotic use of 424 treatments per 1000 inhabitants per year can be compared to prescriptions rates from a Dutch study33 of 45 primary practices with a prescribing rate of 300 treatments per 1000 patient years. The Netherlands is a low antibiotic prescribing country and furthermore out-of-hours use was not included.We did not have data to assess the specific consultation rates for acute respiratory tract infections. As a result, we are unable to compare data on proportion of consultations for acute respiratory tract infection that resulted in an antibiotic prescription.A substantial number of prescriptions were not available for analysis due to either no clinical indication present on the electronic prescriptions or non-specific indications hampering the precise assessment of incidences of specific acute respiratory infections. However, the remaining large number of data can provide a valid specification of the antibiotic use pattern for selected clinical indications, as we did not find evidence of a systematic difference regarding patient or provider age and sex in the overall antibiotic use between scripts with no indications or unspecific indications vs. scripts with specific indications (data not shown).A number of clinical indications may be missing from telephone or handwritten prescriptions. We have no methods to assess this amount, but we believe this number is relatively small and with no substantial impact on the antibiotic use pattern.It may be argued, those GPs who are not up-to-date or who do not want to be part of surveillance systems could be underrepresented or not coding in a meaningful way. The clinical indication functionality on electronic prescriptions is still quite new and not all GPs may find it intuitive, easy to use or relevant. A GP could just use the first indication from the drop down menu throughout the day. This would threaten the validity of the assigned clinical indications and make the antibiotic use pattern less precise. We believe this problem is virtually impossible to eliminate, but future studies should be undertaken to improve understanding of prescriptions with missing and non-specific clinical indications.35Future studies would benefit from differentiating between daytime and out-of-hours antibiotic use as Huibers et al. have shown that out-of-hours antibiotic use was high and often second-line agents were issued, especially among patients >60 years of age.37 A quality indicator is defined as a specific and measurable instrument that can be used to assess the quality of care provided.38 For optimal impact and validity context, specific quality indicators should be developed and implemented. This process is presently ongoing in Denmark. The systematic implementation of such valid quality indicators may be beneficial to future patients by assisting individual clinics striving to improve care as well as to monitor national surveillance targets on antibiotic use.39Assessing rational antibiotic prescribing is challenging as no formal quality indicators have been formally developed and validated in a Danish setting.The number of unspecific indications is high and an effort to reduce this group is warranted, bearing in mind that not all cases issued a prescription may present with a clear site of infection at the time of consultation. Possible changes might include grouping of selected clinical indications, such as lower or upper acute respiratory tract infection, and providing more options for symptoms or complaints in the clinical indications such as cold and cough. To more accurately reflect the clinical indications for which antibiotics are prescribed and thus optimize understanding changes in antibiotic use, a more direct link from medical records into the electronic prescribing system is warranted.This study confirms that penicillin V is the most commonly prescribed antibiotic agent for treatment of patients with an acute respiratory tract infection in Danish general practice. However, the high prescribing rate of second-line agents, like macrolides and extended-spectrum penicillins, could be improved significantly. Future initiatives to promote rational prescribing of antibiotics should address specific clinical indications like AOM, acute rhinosinusitis, and pneumonia.40All Danish citizens who redeemed an antibiotic prescription  between July 1st 2012 to June 31st 2013 were included. This 1-year study period was chosen to ensure a coherent winter season with its corresponding influenza peak.41 This registry contains complete information on all prescriptions redeemed by Danish residents at outpatient pharmacies from January 1st 1995 onwards. Antibiotics are only available by prescription and purchased solely in pharmacies in Denmark. For each redeemed prescription, the registry contains information on the following variables relevant to this study: provider practice identification number (encrypted), type of antibiotic, patient age and gender, as well as clinical indication.National data on use of antibiotic therapy in Denmark was extracted from the Danish National Prescription Database.Clinical indications are provided by the electronic prescription system. As such they are not directly linked to the International Classification of Primary health Care (ICPC-2) coding system used in the GPs medical records. The GP must select an appropriate clinical indication from a pre specified drop down list or enter a free text indication prior to signing of an antibiotic.42Antibiotic agents were classified according to the anatomical therapeutic chemical (ATC) index , down to 5th level; chemical substance.43 contains data on all Danish residents through an individual identifier (encrypted), which allowed us to keep track of all study subjects.The Danish Civil Registration System40 All information on patient and prescriber level was encrypted.General practice providers were identified by the unique identifiers for general practitioners (encrypted), obtained from Statens Serum Institute, to distinguish GP prescriptions from the entire primary care sector, which also includes other medical providers such as ear-nose-throat specialist, dentist etc.Statistikbanken.dk, a service from the Statistics Danmark, was used to determine population size in total and the included age groups.11 on antibiotic use recommended penicillin V as empirical first choice for AOM, acute tonsillitis, acute rhinosinusitis, and pneumonia. In case of penicillin allergy, a macrolide was recommended. The recommendation for AECOPD was co-amoxicillin. In case of penicillin allergy, a tetracycline was recommended. Uncomplicated acute bronchitis is considered a self-limiting disease and no antibiotic is recommended.During the study period (2012–2013), the Danish national guideline44We assumed that recommended empirical first-line treatment should account for at least 80% of prescriptions in order to adhere to guidelines.45No specific guidance regarding diagnostics were included, however a general recommendation regarding use of C-reactive protein was adopted in general practice (the C-reactive protein speedometer).40 <5 years; 5–14 years; 15–44 years; 45–64 years; 65–74 years, 75+ years.A dataset consisting of clinical indications and antibiotic prescriptions was created by merging datasets by their unique practice identification numbers.The following six age groups for each specific indication were used;46 As a result, both upper and lower respiratory tract infections were included. Furthermore we chose to include AOM to the group of respiratory tract infections in primary care. This was due to the fact that the clinical picture of AOM rarely presents as an isolated disease entity, but most often is accompanied by additional common cold symptoms.When assessing the clinical indications for antibiotic prescribing, we chose to include the full range of acute respiratory tract infections; all R codes in the International Classification of Primary Care, second edition.The clinical indicators on the most common acute respiratory tract infections in primary care were grouped and analyzed: AOM, acute tonsillitis, acute rhinosinusitis, acute bronchitis, pneumonia, and AECOPD. Descriptive analyses of antibiotic use were performed, such as percentages, cumulated percentages, no. of treatments per 1000 inhabitants for each acute respiratory tract infection.11 Danish national guidelines on antibiotic use recommends narrow-spectrum penicillin (penicillin V) as first-line agent for most respiratory cases, except AECOPD, where the recommendation in 2012–2013 was co-amoxicillin. All calculations were done in SAS version 9.3 .In addition, the accordance of GPs’ antibiotic management of patients with acute respiratory tract infections was assessed by comparing to national guidelines.The study was approved by the Danish Data Protection Agency (J.nr. 2012-41-0159). According to Danish law, no approval from an ethics committee is required for strictly registry based studies.All relevant data are available from the authors, however Danish law on data protection prohibits access to raw data.