Infections are rare but important causes of stroke. Among these, varicella zoster virus has been known to cause ischemic stroke. During an attack of herpes zoster ophthalmicus, it has been hypothesized that the virus replicates in the trigeminal ganglion and travels via the trigeminal nerve centrally to cause cerebral vasculopathy. Here we present a case of a 69 year-old Caucasian immunocompromised woman who suffered recurrent ischemic infarcts within the same vascular distribution following an episode of zoster ophthalmicus three months prior. An imaging technique termed black-blood magnetic resonance imaging was utilized to aid in the diagnosis of cerebral vasculitis. The case is used to provide a literature review of the pathogenesis, diagnosis, and treatment of cerebral varicella zoster vasculopathy. In situations where an isolated unilateral cerebral vasculopathy is identified, neurologists are urged to consider varicella zoster as a treatable etiologic agent, as untreated vasculopathy can lead to further strokes. We describe a case of varicella zoster virus (VZV) vasculopathy in a 69 year old woman with myasthenia gravis on immunosuppressive therapy who presented with recurrent strokes in the same vascular territory three months after an episode of herpes zoster ophthalmicus. Initial imaging with magnetic resonance imaging (MRI) and magnetic resonance angiogram (MRA) could not distinguish between atherosclerosis and vasculitis. A black-blood magnetic resonance imaging (BB-MRI) demonstrated inflammation in the walls of the vascular territories responsible for the strokes. Isolation of VZV DNA from the cerebral spinal fluid (CSF) and demonstration of vasculitis on the BB-MRI confirmed the presence of VZV vasculopathy.Varicella zoster virus is a human alpha-herpesvirus that causes varicella (chickenpox) with primary infection. Subsequently, the virus becomes latent in cranial nerve and dorsal root ganglia along the neuraxis. As cell-mediated immunity wanes, the virus may reactivate to cause zoster which can lead to various complications, including vasculopathy . While tMackenzie et al.  describeA 69 year-old woman with a history of myasthenia gravis on immunosuppressant therapy (mycophenolate 1000 mg BID and prednisone 5 mg QD) for the previous two years presented to our facility with acute left-sided weakness and numbness. She reported two recent ischemic stroke hospitalizations over the preceding 3 months and that she was fully compliant with her ongoing post-stroke medical management. Initial non-contrast computed tomography (CT) of the head was negative for hemorrhage or evidence of acute ischemia, but did demonstrate regions consistent with her prior infarcts in the right fronto-parietal regions. Intravenous thrombolytic therapy was not administered secondary to timing and improving symptoms. National Institutes of Health Stroke Scale (NIHSS) score was 8 with left-sided hemiparesis and paresthesias involving face, arm, and leg.Further review of her prior history revealed that approximately 3 months before her current presentation she had experienced acute onset of left lower extremity weakness with imaging demonstrating scattered ischemic infarcts in the right frontal lobe. At that time, aspirin and statin therapy was initiated and she was transferred to a rehabilitation facility. Two weeks later, while at the rehabilitation facility, she experienced acute left facial droop and left upper extremity weakness with no sensory loss or neglect with imaging demonstrating new scattered ischemic infarcts involving the right fronto-parietal region and corona radiata. Computed tomography angiogram (CTA) of the head demonstrated marked irregularity of the right A1 segment of the anterior cerebral artery (ACA). There was also calcification in various bilateral cerebral vessels without flow-limiting stenosis. Intracranial atherosclerosis was hypothesized as the likely culprit of her recurrent strokes and dual anti-platelet therapy with aspirin and clopidogrel was initiated; she continued on her statin. After a short course of rehabilitation she was discharged home. She then presented to our facility approximately three months after the initial stroke.During her hospitalization in our facility, work-up revealed a hemoglobin A1C of 4.7% and a LDL of 39 mg/dL. Transthoracic echocardiogram showed a preserved ejection fraction with moderate calcified aortic stenosis, no patent foramen ovale (PFO), and no atrial thrombus. EKG and telemetry demonstrated normal sinus rhythm. MRI demonstrated new acute scattered ischemic infarctions involving the right corona radiata and right posterior limb of internal capsule . 3D TimeTo differentiate between vasculitis and atherosclerosis causing stenosis, intracranial arterial wall imaging (black blood MRI) was performed using a 3T MR scanner. The arterial wall imaging protocol consisted of pre- and post-contrasted axial and coronal T1-weighted images with inversion recovery, BB-MRI demonstrated circumferential wall enhancement of the supraclinoid portion of right internal carotid artery, A1 and proximal A2 segments of the right ACA, and M1 and proximal M2 segments of the right MCA, compatible with vasculitis . SubsequThe presence of VZV DNA in CSF and the pattern of vessel wall enhancement on the BB-MRI supported the clinical diagnosis of VSV vasculopathy, as such a digital subtraction angiogram (DSA) was not deemed necessary. With guidance from our Infectious Disease Consult Service, the patient was started on treatment with intravenous acyclovir (10 mg/kg in three daily doses) for total of 2 weeks, then switched to valacyclovir 500 mg BID as suppressive therapy in light of her continued MG immunosuppression therapy requirements. Following inpatient hospitalization, the patient was transferred to acute stroke rehabilitation and is now followed in our outpatient stroke clinic. At her most recent evaluation approximately 5 months after discharge, she reported no new neurological symptoms and improving residual deficits; her most recent examination was positive for left upper extremity pronator drift, 4/5 strength in left lower extremity, and diminished pain and temperature on the left side. She is ambulating with use of a rolling walker.Herpes zoster has been shown to be associated with an increased incidence of stroke in large population-based studies -13. SreeLangan et al. demonstrated that patients treated for herpes zoster had a lower incidence of stroke than untreated patients . InteresThe clinical features of VZV vasculopathy have been described as protean . PatientIn a review of 30 patients with confirmed VZV vasculopathy, 67% had CSF pleocytosis and 97% patients had abnormal findings on brain imaging, usually at the gray-white matter junction . Only 13Varicella virus is the only known human virus to have been isolated from vessels responsible for cerebral ischemia or infarction . VasculoVascular remodeling occurs with the development of a neo-intima composed of smooth muscle cells that may have originated from the tunica media , a proceIt is important to note that the immunosuppressed patient described in this case report developed VZV vasculopathy despite having been treated with oral antivirals when diagnosed with herpes zoster ophthalmicus. This suggests that host immunity may also play a role in determining the progression from herpes zoster to VZV vasculopathy. Further, it remains unclear from population based studies of stroke post herpes zoster and isolated cases of VZV vasculopathy if similar pathophysiological mechanisms are involved in both processes or if they represent a range of infection-triggered inflammatory response that is modulated by host immunity.As described above, imaging studies using BB-MRI and CSF evaluations for VZV-specific IgG and VZV DNA were central to our diagnosis. It is important to note that not all cases of VZV vasculopathy demonstrate angiographic abnormalities using traditional angiographic imaging techniques; moreover, if present, vasculopathy may also be difficult to discern from ongoing atherosclerotic disease. Nagel et al. document that 30% of patients with VZV vasculopathy do not have angiographic findings on MRI or CT . Obusez There are no randomized studies defining or comparing guidelines for the treatment of varicella zoster vasculopathy in immunocompromised or immunocompetent patients. In one study of 30 patients with VZV vasculopathy, 15 patients were treated with acyclovir alone of which 9 improved, 1 stabilized, and 5 worsened (3 of which improved with steroids). Of the 12 patients treated with steroids and acyclovir, 8 improved, 1 remained stable and 3 worsened .The roles of antiviral drugs or VZV vaccines in stroke risk reduction from either primary or reactivated varicella virus infection are unknown. The optimal therapy for treatment of VZV vasculopathy for either immunocompetent or immunocompromised patients is also unknown and requires further research. Shown in VZV vasculopathy should be considered in the setting of recent shingles infections, particularly among those with eye involvement and/or among patients that are immunocompromised. As described in our case, we were able to document isolated vessel wall inflammation through the use of BB-MRI that was not seen on traditional MRA, and that enhanced atypically as compared with atherosclerotic disease. We believe our case is among the first in the literature utilizing this imaging technique for real-time clinical decision-making to demonstrate vessel wall inflammation in a patient with VZV vasculopathy. As such, we propose BB-MRI as a methodology to screen patients presenting with acute stroke in the setting of a recent herpes zoster infection, as such patients would benefit from treatment with antiviral therapy. Combined with the detection of VZV IgG and/or VZV DNA from the CSF, this technique may improve the identification of patients with VZV vasculopathy and possibly demonstrate response to therapy. Further studies implementing BBMRI may also determine what percentage of strokes following herpes zoster are due to vasculitis and provide further insight into the pathophysiology of VZV vasculopathy. A review of the literature also indicates that antiviral +/− corticosteroid therapy results in 75-80% stabilization or improvement in neurologic findings in immunocompromised adults with VZV vasculopathy.


Biomarkers allowing the characterization of malignancy and therapy response of oral squamous cell carcinomas (OSCC) or other types of carcinomas are still outstanding. The biochemical suicide molecule endonuclease DNaseX (DNaseI-like 1) has been used to identify the Apo10 protein epitope that marks tumor cells with abnormal apoptosis and proliferation. The transketolase-like protein 1 (TKTL1) represents the enzymatic basis for an anaerobic glucose metabolism even in the presence of oxygen (aerobic glycolysis/Warburg effect), which is concomitant with a more malignant phenotype due to invasive growth/metastasis and resistance to radical and apoptosis inducing therapies.Expression of Apo10 and TKTL1 was analysed retrospectively in OSCC specimen (n = 161) by immunohistochemistry. Both markers represent independent markers for poor survival. Furthermore Apo10 and TKTL1 have been used prospectively for epitope detection in monocytes (EDIM)-blood test in patients with OSCC (n = 50), breast cancer (n = 48), prostate cancer (n = 115), and blood donors/controls (n = 74).Positive Apo10 and TKTL1 expression were associated with recurrence of the tumor. Multivariate analysis demonstrated Apo10 and TKTL1 expression as an independent prognostic factor for reduced tumor-specific survival. Apo10+/TKTL1+ subgroup showed the worst disease-free survival rate in OSCC.EDIM-Apo10 and EDIM-TKTL1 blood tests allowed a sensitive and specific detection of patients with OSCC, breast cancer and prostate cancer before surgery and in after care. A combined score of Apo10+/TKTL1+ led to a sensitivity of 95.8% and a specificity of 97.3% for the detection of carcinomas independent of the tumor entity.The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 allows a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence. The immunohistochemical detection of biomarkers in tumor tissue-sections is an essential and powerful technique to determine the malignancy of the tumor and to stratify cancer patient treatment . The sucIn order to establish biomarkers applicable to all tumor entities, biomarkers for two fundamental biophysical mechanisms in mammalian cells have been selected. Despite the extreme complexity of signaling processes within and between cells, only a few principle biophysical mechanisms are known to determine the existence and death of mammalian cells.One important biophysical mechanism which determines the fate and death of a cell is the cleavage of nuclear DNA by endonucleases . InhibitAnother important, if not the most important biophysical mechanism of life is the way of energy release within cells. Multicellular organisms depend on energy release either by fermentation or by oxidative phosphorylation (OxPhos). Therefore, only two ways of energy release are possible . While fThe Apo10 protein epitope is detected by the monoclonal antibody Apo10, which has been raised against a DNaseX peptide sequence. DNaseX is a member of the DNaseI-protein family consisting of DNaseI, DNaseX (DNaseI-like 1), DNaseI-like 2 and DNaseI-like 3 (DNase gamma). The Apo10 epitope is present in tumor cells and in very few non-malignant cell types ,29.Our study describes the immunohistochemical evaluation of the two biomarkers Apo10 and TKTL1 for characterization of OSCC tissue sections. Furthermore, both biomarkers have been detected intracellularly in monocytes using the epitope detection in monocytes (EDIM) technique, allowing a sensitive and specific noninvasive detection of OSCC, breast and prostate cancer patients by blood samples. This new blood test is based on the EDIM technology ,29-31, wIn the present study, we analysed retrospectively the potential prognostic and predictive influence of Apo10 and TKTL1 expression on clinicopathological parameters and on disease-free survival rates in a large patient cohort with OSCC. In addition to the retrospectively assessed Apo10 and TKTL1 data, prospectively Apo10 and TKTL1 have been determined using the EDIM technique. EDIM-Apo10 and EDIM-TKTL1 blood test was performed in patients with primary and/or recurrent OSCC, breast cancer patients, prostate cancer patients and healthy individuals (blood donors).We retrospectively reviewed the records of 161 patients after primary radical R0 tumor resection in our department over a period of ten years and healthy individuals . The material had been stored and was investigated with permission of the patients and the local ethics committee . Patient selection criteria and routine histopathological work-up are described as recently published . Tumor bFor immunohistochemical analysis, two anti-DNaseX  monoclonal antibodies have been used: Apo10  and ab54750 . Furthermore, monoclonal anti-TKTL1 antibody  were analysed for Apo10 and TKTL1 expression in the tumor tissue and averaged in each case. The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tissue area, was semi-quantitatively scored on a scale of 0 to 3 as the following: 0, <10%; 1, 10–30%; 2, 30–60%; 3, >60%. The intensities of the signals were scored as 1+, 2+, and 3+. Then, a combined score (0–9) for each specimen was calculated by multiplying the values of these two categories [Five representative chosen high power fields (1 HPF = 0.237 mmtegories . Cases whttp://rsb.info.nih.gov/ij/) coupled with immunomembrane plug-in (http://153.1.200.58:8080/immunomembrane/) was used to assess the quantification of TKTL1 immunoreactivity in microscopically acquired JPEG images of OSCC samples. Staining completeness (0–10 points) and intensity (0–10 points) were added for a combined score (0–20 points) [http://153.1.200.58:8080/immunoratio/). The results were expressed as percentages [Moreover, for computer-assisted semi-quantitative analysis of TKTL1 expression, ImageJ software ( points) . Cases wcentages . From ApThe sequential double staining (co-expression) was analysed for Apo10 with TKTL1. The secondary antibody used for IHC/ICC double staining for Apo10 was an alkaline phosphatase (AP)-conjugated AffiniPure donkey-anti-rat IgG (Jackson ImmunoResearch), used at 1:200 dilution. The secondary antibody of TKTL1 was a horseradish peroxidise (HRP)-conjugated AffiniPure donkey-anti-mouse IgG, used at 1:200 dilution (Jackson ImmunoResearch).For IHC and ICC double staining of Apo10 and TKTL1, slides were incubated with the primary Apo10 antibody or control antibody overnight at 4°C in a humidified chamber and with secondary AP-conjugated antibody for 30 minutes at room temperature in a humidified chamber followed by 20 minutes of incubation with Fast Red . Subsequently, the slides were incubated with the second primary TKTL1 antibody or control antibody overnight at 4°C in a humidified chamber and with secondary HRP-conjugated antibody for 30 minutes at room temperature in a humidified chamber followed by 5 minutes of incubation with 3,3′-Diaminobenzidine .Slides were counterstained with Haemalaun and mounted with Glycergel . The photographed images were imported into the Microsoft Office Picture Manager.4) from the OSCC cell lines BICR3 [2.We analysed Apo10 and TKTL1 expression in cells  with primary and/or recurrent OSCC as described previously by flow cytometric analysis ,29. In tInformed consent to participate was obtained for the blood tests collected prospectively from patients and volunteers .Samples were analyzed with a BD FACSCantoII . At least 10,000 relevant events were collected for each sample. FITC, PE, PerCP and APC signals were recorded as logarithmically amplified data. Analysis was performed using BD FACSDiva software v6.1. . The result of the EDIM test is given as a relative score indicating the relative amount of CD14/CD16 positive monocytes harbouring Apo10 (or TKTL1) compared to the total amount of CD14/CD16 positive monocytes multiplied with 10. For example, a Apo10 score of 139 means that 13.9% of CD14/CD16 positive monocytes harboured Apo10 intracellularly . The resχ2) and Fisher’s exact test were used to investigate the relation between two categorical variables. Non-parametric Kendall’s tau (т) correlation coefficient was measured to assess the accuracy  between the two quantification methods of immunohistochemical analysis. All p-values presented were 2-sided and p < 0.05 was considered statistically significant.Statistical analysis was performed with MedCalc Software, Version 12.7.0 . Disease-free survival (DFS) time was calculated from the time of tumor resection until appearance of obvious locoregional recurrence or tumor conditional death, respectively. The DFS times were estimated using the Kaplan-Meier method  and wereTo analyse differences in the EDIM-Apo10 and EDIM-TKTL1 scores among healthy individuals (blood donors) and cancer patients, Receiver Operating Characteristics (ROC) analysis was performed . ROC anaA preliminary study was carried out to assess the accuracy between the two quantification methods of immunohistochemical analysis. There were significant correlations between the first (observer related semi-quantitative scoring) and the second (computer-assisted (semi)-quantitative analysis) assessment. Apo10 expression: т = 0.868, p < 0.0001 and TKTL1 expression: т = 0.886, p < 0.0001.Two monoclonal antibodies raised against DNaseX have been selected for expression analysis of OSCC tumors. Antibody ab54750 shows a cytoplasmic and a focal nuclear staining pattern in tumor and in stromal cells, respectively . Table To analyse survival rates in patients after successful (R0) curative surgical resection of OSCC, patients were divided in positive and negative Apo10 expressors (dichotomous variables) measured by observer related semi-quantitative scoring.In our study population, cervical lymph node metastasis  and extracapsular spreading of lymph nodes (p < 0.0001) were shown to be unfavorable factors in univariate analysis of all (n = 161) OSCCs. Tumor size  and grading  were not found to be unfavorable factors in univariate analysis.To analyse differences in tumor related survival dependent on Apo10 expression in OSCC, we divided the patients in two subgroups as described above. Survival in subgroup with positive Apo10 expression (Apo10+) in OSCCs was significantly worse in comparison to the subgroup of patients lacking Apo10 expression. Apo10+: n = 132, p = 0.0027, HR = 6.4509  OSCCs  curative surgical resection of OSCC, patients were divided in positive and negative TKTL1 expressors (dichotomous variables) measured by observer related semi-quantitative scoring.To analyse differences in tumor related survival dependent on TKTL1 expression in OSCC, we divided the patients in two subgroups as described above. Survival in subgroup with positive TKTL1 expression (TKTL1+) in OSCCs was significantly shorter in comparison to the subgroup of patients lacking TKTL1 expression. TKTL1+: n = 68, p < 0.0001, HR = 3.8382  as independent prognostic factors in all (n = 161) OSCCs . 71 of 74 healthy individuals were negative using the combined score (Table In 32 out of 50 OSCC patients (n = 32/50), Apo10 and TKTL1 expression had been analysed by immunohistochemistry, since in 18 cases (n = 18/50) biopsy was not possible due to palliative treatment indications in recurrent OSCC or due to biopsy conducted by external surgeons. 29 out of 32 OSCCs were positively stained for Apo10  and 30 out of 32 individuals were positive for TKTL1 . Measurement of EDIM-Apo10 and EDIM-TKTL1 revealed normal scoring levels after R0 resection and convalescence  alone as definitive therapy, whereas 50 (31%) patients had adjuvant radiotherapy with/without chemotherapy. Adjuvant treatment in association with UICC stages is shown in Additional file 11 alone In 1991 Nobel laureate Professor Harald zur Hausen initiated a genome analysis program in the German Cancer Center focusing on the genomic region Xq28. At that time there was no evidence for the presence of cancer related genes in this region except the already known MAGE genes. This genomic region was systematically cloned in cosmids, ordered in contigs and further analysed for the presence of genes, conserved between human and pig, encoding tissue-specific expressed transcripts . As a reTo analyse the expression of DNaseX protein in OSCC, a DNaseX peptid has been used to generate monoclonal antibody Apo10. Although another commercially available anti-DNaseX monoclonal antibody revealed an overexpression of the detected protein in OSCC, its staining pattern is distinct from Apo10 antibody and less tumor specific. This difference in staining patterns of both antibodies could be due to differences in crossreactivity to other DNaseI protein family members or even unrelated proteins, but could also be the consequence of epitope masking by protein binding. Such selective epitope detection by monoclonal antibodies has been revealed for DNase-gamma (DNaseI-like 3) . Two monSimilar to the DNase-gamma peptide, the DNaseX peptide may represent an epitope (Apo10 epitope) which is the target of molecular and/or biochemical change(s) leading to differential accession by monoclonal antibodies. Monoclonal antibody Apo10 detects a protein epitope present in the nucleus of apoptotic benign cells, but also in the nucleus of tumor cells. Whereas benign apoptotic cells (e.g. heart muscle cells of myocarditis patients) execute apoptosis, tumor cells apparently block the endonuclease driven execution of apoptosis by expression of proteins inhibiting endonucleases ,5 or othThe Apo10 epitope is present in neoplastic cells including carcinomas, sarcomas, glioblastomas, lymphomas, and leukemias , whereasAlthough the overexpression of TKTL1 takes also place in carcinomas , sarcomaThe clinical relevance of these two fundamental biophysical processes is underlined by the fact, that Apo10 and TKTL1 presence in tumors represent two independent processes which are associated with advanced tumor stages and reduced tumor-specific survival in OSCC. This allows for the first time a two-step model of detection and characterization of tumors based on two biomarkers. OSCC tumors negative for Apo10 and TKTL1 represented tumors with favourably prognostic impact on survival, Apo10 presence indicated malignant OSCC tumors, and Apo10+/TKTL1+ OSCC tumors turned out to be more malignant tumors with an invasive/metastasing phenotype and worst prognostic impact on survival.A large number of studies underlined the clinical relevance of increased TKTL1 expression on gene, transcript and protein level, since TKTL1 expression correlates with poor patient outcome and metastasis in many solid tumors -27. InhiA multistep process comprising initially six biological capabilities has been proposed as the basis for development of human tumors. The so called hallmarks of cancer include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis . Our ApoIn a proof of concept study the biomarkers Apo10 and TKTL1 have been used exploiting the epitope detection in monocytes (EDIM) technology. The EDIM technology allows a noninvasive detection of tumor proteins in blood. EDIM-Apo10 and EDIM-TKTL1 blood tests have been prospectively conducted in patients with primary or recurrent OSCC as well as in patients with primary breast and prostate cancer. In patients with histologically confirmed OSCC, breast and prostate cancers, blood samples before surgery revealed significant elevated levels of Apo10 and TKTL1 in CD14/CD16 positive monocytes compared to blood donors. By using a single cut-off for all three tumor entities it was possible to identify cancer patients by either EDIM-Apo10 or EDIM-TKTL1 blood test in a specific and sensitive manner. The combination of EDIM-TKTL1 and EDIM-Apo10 scores increased the sensitivity to 95.8% and the specificity to 97.3% in all cancer samples/entities.The combined detection of two independent fundamental biophysical processes by the two biomarkers Apo10 and TKTL1 may allow a sensitive and specific detection of neoplasia in a noninvasive and cost-effective way. Further prospective trials are warranted to validate this new concept for the diagnosis of neoplasia and tumor recurrence.OF, HH and JFC are employees and shareholders of TAVARLIN AG, Pfungstadt, Germany and declare a potential conflict of interest due to the possible utilization of Apo10/DNaseX and TKTL1 for diagnostic and/or therapeutic purposes. The authors have no other affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.MG and JFC conceived the study, carried out immunohistochemistry studies, performed the statistical analyses, and drafted the manuscript. OF, SS and PT performed flow cytometric analysis. TB and JS analysed histopathological specimen and carried out immunohistochemistry studies. AS, JH, HJM, and KK carried out the data collection. AM and TN performed cell culture experiments. OF, HH, and SR participated in the design of the study and coordination and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/569/prepubDNaseX staining. Immunohistochemistry shows representative images of antibody ab54750 staining  compared to Apo10 staining . Antibody ab54750 shows cytoplasmic and a focal nuclear staining pattern, whereas Apo10 is detected exclusively in the nucleus. The blue color shows the nuclear counterstaining by hematoxylin. The square box demonstrates the area of interest  which is also shown in larger magnification .Click here for fileDNaseX (Apo10) staining in human normal oral squamous epithelial cells. Immunohistochemistry shows representative image of Apo10 staining in human normal oral squamous epithelial cells. Apo10 is not detected in human normal oral squamous epithelial cells. The blue color shows the nuclear counterstaining by hematoxylin. Original magnification: ×200-fold.Click here for fileSurvival curve of OSCC patient subgroup analysis measured by Apo10/TKTL1 co-expression. Kaplan-Meier survival curves for DFS stratified by Apo10-/TKTL1- (blue line), Apo10+/TKTL1- (red line), Apo10-/TKTL1+ (grey line), and Apo10+/TKTL1+ (green line) subgroups (a). Compared with Apo10+/TKTL1- (red line), Apo10+/TKTL1+ (green line) subgroup shows the worst DFS . The most favorable prognosis is demonstrated by the Apo10-/TKTL1- (blue line) subgroup.Click here for fileDNaseX (Apo10) staining in benign cells of the myocardium and two different human epithelial tumor entities - carcinomas of the lung, and colon. Hematoxylin and eosin stain (H&E) shows myocardium (a) and different types of carcinomas . Immunohistochemistry shows representative images of Apo10  in human apoptotic  benign cells of a patient after myocarditis and in carcinomas of the lung, and colon, which is detected in the nucleus. Apoptotic cells (Caspase-3 cleaved) are increased in benign tissue (i) compared with decreased detection of apoptotic cells in carcinomas . Both, benign and malign tissue types stained Apo10+. The blue color shows the nuclear counterstaining by hematoxylin. Original magnification: ×200-fold. AC, adenocarcinoma; SCC, Squamous cell carcinoma.Click here for fileDNaseX (Apo10) staining in bladder and breast carcinoma. Hematoxylin and eosin stain (H&E) shows bladder and breast carcinomas . Immunohistochemistry shows representative images of Apo10  in human apoptotic  cells in carcinomas of the bladder and mammary gland (breast), which is detected in the nucleus. Apoptotic cells (Caspase-3 cleaved) in carcinomas  are decreased compared with benign tissue  and TKTL1 immunocytochemical staining in BICR3, BICR56, and SCC-4 OSCC cell lines. IgG control shows no staining . Images show representative immunocytochemical staining of Apo10 , and TKTL1 . The blue color shows the nuclear counterstaining by hematoxylin. Original magnification: ×400-fold.Click here for fileFlow cytometric analysis of Apo10+, TKTL1+ cancer cells and immunocytochemical Apo10+/TKTL1+ double staining. Flow cytometric analysis shows representative Apo10 (a) and TKTL1 (b) labeling in BICR56 cancer cells as a positive control. FITC, Fluoresceinisothiocyanate. Immunocytochemical staining shows a representative image of Apo10+/TKTL1+ (c) tumor cells in BICR56 OSCC cell line. The red nuclear chromogen color (Fast Red) indicates positive Apo10 staining and the brown cytoplasmic chromogen color (DAB) indicates positive TKTL1 staining (arrows). Asterisks show single Apo10 (red) or TKTL1 (brown) positive cells. Original magnification: ×400-fold.Click here for fileImmunohistochemical Apo10+/TKTL1+ double staining. Immunohistochemical Apo10+/TKTL1+ double staining of a representative double positive OSCC tissue shows nuclear Apo10+  and cytoplasmic TKTL1+  co-expression. The square box demonstrates area of interest , which is also shown in a larger magnification .Click here for fileReceiver Operating Characteristics (ROC) analysis of combined EDIM Apo10/TKTL1 score in OSCC (n = 50) compared with healthy individuals (n = 74), and interactive dot diagrams. The true positive rates (sensitivity) are plotted in function of the false positive rate (100-specificity) for measurement of the cut-off point: ROC analysis for the diagnosis of primary or recurrent OSCC shows calculated cut-off value with highest diagnostic accuracy (arrows) of combined EDIM Apo10/TKTL1 (a) score combined EDIM-Apo10 plus EDIM-TKTL1 score >227: sensitivity 94.0%, 95% CI 83.5–98.7%, specificity 97.3%, 95% CI 90.6–99.7%). Dotted lines show 95% CI. OSCC, oral squamous cell carcinoma. In the interactive dot diagrams  the data of healthy controls and OSCC group are displayed as dots on two vertical axes. The horizontal line indicates the cut-off points with the best separation/highest accuracy  between healthy controls and OSCC group. The corresponding test characteristics sensitivity and specificity are shown above.Click here for filePre- and postoperative epitope detection in monocytes (EDIM)-Apo10 and TKTL1 scores in patients with oral squamous cell carcinoma (n = 3).Click here for filePre- and postoperative epitope detection in monocytes (EDIM)-Apo10 and TKTL1 scores in patients with breast cancer (n = 3).Click here for filePre- and postoperative epitope detection in monocytes (EDIM)-Apo10 and TKTL1 scores in patients with prostate cancer (n = 6).Click here for fileReceiver Operating Characteristics (ROC) analysis of EDIM-Apo10, EDIM-TKTL1, and combined EDIM Apo10/TKTL1 score in all cancer samples  compared with healthy individuals (n = 74). The true positive rates (sensitivity) are plotted in function of the false positive rate (100-specificity) for measurement of the cut-off point: ROC analysis for the diagnosis of all cancer samples/entities  shows calculated cut-off value with highest diagnostic accuracy (arrows) of EDIM-Apo10 (a), EDIM-TKTL1 (b), and combined EDIM Apo10/TKTL1 (c) score . Dotted lines show 95% CI. OSCC, oral squamous cell carcinoma; BC, breast cancer; PC, prostate cancer. In the interactive dot diagrams  the data of healthy controls and cancer group are displayed as dots on two vertical axes. The horizontal line indicates the cut-off points with the best separation/highest accuracy  between healthy controls and cancer group. The corresponding test characteristics sensitivity and specificity are shown above.Click here for fileAdjuvant treatment of 161 patients with OSCC according to UICC stages.Click here for file


Hyponatremia (sodium levels of <135 mEq/L) is one of the most common electrolyte imbalances in clinical practice, especially in patients with neurologic diseases. Hyponatremia can cause cerebral edema and brain herniation; therefore, prompt diagnosis and proper treatment is important in preventing morbidity and mortality. Among various causes of hyponatremia, diagnosing syndrome of inappropriate antidiuretic hormone secretion (SIADH) and cerebral/renal salt wasting syndrome (C/RSW) is difficult due to many similarities. SIADH is caused by excess of renal water reabsorption through inappropriate antidiuretic hormone secretion, and fluid restriction is the treatment of choice. On the other hand, C/RSW is caused by natriuresis, which is followed by volume depletion and negative sodium balance and replacement of water and sodium is the mainstay of treatment. Determinating volume status in hyponatremic patients is the key point in differential between SIADH and C/RSW. However, in most situations, differential diagnosis of these two diseases is difficult because they overlap in many clinical and laboratory aspects, especially to assess differences in volume status of these patients. Although distinction between the SIADH and C/RSW is difficult, improvement of hypouricemia and an increased fractional excretion of uric acid after the correction of hyponatremia in SIADH, not in C/RSW, may be one of the helpful points in discriminating the two diseases. In this review, we compare these two diseases regarding the pathophysiologic mechanisms, diagnosis, and therapeutic point of view. Hyponatremia, defined by sodium levels of <135 mEq/L, is one of the common electrolyte imbalances in clinical practice, especially among patients with neurologic diseases . Acute hAmong various causes of hyponatremia, the diagnoses of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) and cerebral/renal salt wasting syndrome (C/RSW) are still confusing due to many similarities. According to one retrospective study of electrolyte disturbances in 195 children with acute central nervous system (CNS) diseases, 20 (10.3%) children showed hyponatremia, 7 of them were diagnosed with SIADH, and the other 9 with cerebral salt wasting syndrome (CSW)  secretion . Before C/RSW is another important disease category causing hyponatremia, characterized by volume depletion and negative sodium balance. Peters et al. first described C/RSW in a report of three patients with neurogenic diseases and hyponatremia . FollowiThe pathophysiology and mechanisms of C/RSW are not completely understood, nevertheless, there is general agreement that the kidneys are unable to conserve sodium leading to variable degrees of reduction in extracellular volume (ECV) depending the extent of the defect in sodium transport and salt intake. In addition, the patient must also have a normally functioning kidney and hypothalamic–pituitary–adrenal axis in order to be diagnosed as C/RSW .This syndrome could be explained with two main hypotheses , 24, 25:If ECV is decreased due to C/RSW, the baroreceptors are triggered and ADH is secreted to restore intravascular volume. The stimulus for ADH secretion by the reduced ECV is more potent than the osmolar effect on ADH secretion, so the patient remains hyponatremic despite the hypoosmolality. This defines the appropriateness of ADH secretion in C/RSW and why saline can remove the volume stimulus for ADH secretion and allow the hypoosmolality to inhibit ADH secretion, increase free water excretion and correct the hyponatremia.Distinguishing between SIADH and C/RSW is important since the proper treatment for each disease is different, and erroneous diagnosis could endanger the patient. However, it is difficult to distinguish the two diseases because many clinical symptoms and laboratory findings overlap. Hyponatremia is a common finding of both SIADH and C/RSW, but the most important difference between the two diseases is the patient’s volume status. SIADH tends to be euvolemic or slightly hypervolemic, whereas C/RSW tends to be hypovolemic , 24. PatIn addition, urine electrolytes can be used to evaluate volume status , 32. GenAlthough laboratory findings such as elevated hematocrit, plasma blood urea nitrogen (BUN), or albumin level might reflect low effective blood volume in C/RSW , 33, 35,Although not tested in children, one recent report demonstrated that bioimpedance spectroscopy showed a higher level of agreement with clinical body fluid estimation than physical examination in adults with hyponatremia, suggesting that bioimpedance spectroscopy could replace physical examination for estimating body fluid status in hyponatremia . BecauseBecause hyponatremia is poorly tolerated in patients with C/RSW diseases, and even a small decrease in serum sodium can aggravate vasogenic cerebral edema , it shouThree percent NaCl should be administered for acute symptomatic hyponatremia. Excessive correction of chronic hyponatremia should be avoided as it can result in osmotic demyelination , 47. ThoSimple fluid restriction is recommended for the treatment of hyponatremia in uncomplicated SIADH , 49. IsoThe treatment purpose of C/RSW is to restore decreased serum sodium levels and intravascular volume due to natriuresis and diuresis and normal saline is frequently used as an initial fluid , 30, 59.Because SIADH and C/RSW have many similarities, differential diagnosis is very difficult in patients with hyponatremia, especially in those with CNS diseases. Nevertheless, it is also important to distinguish between SIADH and C/RSW since the treatment of them differ in many aspects. Volume status of the patients, though it may be difficult to evaluate in the clinical setting, serum uric acid levels and FEUrate after correcting hyponatremia, and the degree of fractional excretions of sodium may be useful in differentiating between the two diseases. Therefore, further studies regarding pathophysiologic mechanisms of diseases and development of diagnostic tools to determine exact volume status is necessary in the future.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RECIST criteria use changes in tumour diameters to quantitatively obtain response assessments for traditional chemotherapeutic cancer treatments: Positive responses are identified by reductions in tumour size while tumour growth signifies non-response.transarterial chemo embolization (TACE) can be used alone or in combination. Local treatment, radiation therapy, immunotherapy and targeted therapy can each result in an increased tumour diameter in spite of treatment response at the cellular level and increased survival times. As a result, tumour diameter can no longer stand alone when assessing oncological treatment strategiesTraditional chemotherapies work primarily by damaging cells that are undergoing rapid growth and division. Immunotherapies work by stimulating the immune system to reject and destroy tumour cells. Targeted cancer therapies block the growth of cancer cells by interfering with specific molecules needed for tumour growth and carcinogenesis. These oncological treatments as well as stereotactic radiation therapy and local treatments, such as image-guided ablations and Tumour heterogeneity and contrast enhancement often change during treatment, and tumour enhancement combined with tumour size has been shown to be successful in assessing treatment response in gastrointestinal stromal tumours (CHOI criteria). Modified CHOI criteria could potentially be used for monitoring the effect of oncological treatment strategies in metastatic renal cell cancer and hepatocellular carcinoma.Dynamic contrast-enhanced imaging  uses changes in tumour enhancement following the intravenous injection of a contrast media to create a time-versus-signal curve. From such curves, reliable quantitative estimates of blood flow, blood volume and permeability can be obtained. Diffusion-weighted MRI and PET can also be used to obtain quantitative measures of physiological processes. A major advantage of DWI-MRI and PET is the possibility of whole body coverage, while DCE imaging is limited to a single tumour or a comparatively small region.Although quantitative assessments of oncological treatment response using physiological parameters have been shown to be superior to size-based response in several studies, the methods mentioned are not yet standardized, and randomized multicentre studies have not yet been performed. When planning such studies, it is important to note that clinical endpoints such as progression free survival and overall survival are superior to morphological imaging results.In the routine assessment of oncological treatment responses, it is becoming increasingly important for the radiologists to know exactly what kind(s) of treatment that the patient has received. Simply reporting changes in tumour size using RECIST is no longer sufficient considering the large percentage of patients who receive advanced and often combined treatment regimens.


The funding statement for this article should read as follows:http://www.esf.org/) and the KU Leuven (www.kuleuven.be) for the IDO-BioCo3 project and KU Leuven Excellence Center project PF/2010/007. EDG acknowledges the KU Leuven for the Grant F+/11/033. MD acknowledges the Fonds de la Recherche Scientifique—FNRS (F.R.S.–FNRS—www.fnrs.be) and the European Research Council (ERC—erc.europa.eu) ERC Advanced Grant “E-SWARM: Engineering Swarm Intelligence Systems” (grant 246939). AET acknowledges the Scientific and Technological Research Council of Turkey (Tubitak—www.tubitak.gov.tr/en) grant TUBITAK-2219. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.”“EF acknowledges the Fund for Scientific Research (FWO)–Flanders (grant 12N7515N). EF, AET and TW acknowledge the European Science Foundation "H2Swarm" program (


Nowadays, nanoparticle probes have received extensive attention largely due to its potential biomedical applications in structural, functional, and molecular imaging. In addition, photoacoustic tomography (PAT), a method based on the photoacoustic effect, is widely recognized as a robust modality to evaluate the structure and function of biological tissues with high optical contrast and high acoustic resolution. The combination of PAT with nanoparticle probes holds promises for detecting and imaging diseased tissues or monitoring their treatments with high sensitivity. This review will introduce the recent advances in the emerging field of nanoparticle probes and their preclinical applications in PAT, as well as relevant perspectives on future development. PAT, as an emerging powerful modality, has the capability to image the structural and functional information of biological tissues with high resolution and satisfactory imaging depth –4. In PAPAT based on intrinsic contrast is dominated by the endogenous agents of biological tissues such as hemoglobin, melanin, and water . As a reIn recent years nanoparticle probes have tremendously impacted scientific researches in biomedical imaging, biological diagnostics, and disease treatments . In compTo date, different types of nanoparticle probes, including metal  nanoparticles, carbon nanotubes, and superparamagnetic iron oxide nanoparticles as well as semiconductor quantum dots, have been synthesized and adopted for different biomedical applications –24. PAT Metal nanoparticles have been employed for photoacoustic molecular tomography because of the biocompatibility, easy modification for targeting, minimized toxicity, and localized surface plasmon resonance peak as well as enhanced optical signals in near-infrared spectral regions –27. In aNanoshells, particularly gold nanoshells, have been demonstrated as valuable vehicles for ex vivo and preclinical studies. Nanoshells coated with polyethylene glycol (PEG) can improve the biocompatibility and the circulating lifetime though the animal body. Nanoshells have been used as an intravascular contrast agent for optical coherence tomography (OCT) and PAT to identify the vascular structures of the brains . NanosheSingle-walled carbon nanotubes have been extensively investigated and used as nanoparticle probes for photoacoustic molecular tomography , 43–45. Superparamagnetic iron oxide nanoparticles (SPIONs) are FDA approved nanoprobes for MRI, which can also be used for nanoparticles probes for photoacoustic molecular tomography due to their perfect biosafety profiles , 46–49. Quantum dots (Qdots) generally refer to semiconductor nanocrystals when the physical dimensions are much smaller than the exciton Bohr radius , 51. Unl2+ ions, seldom changes with their morphologies . Th. Th4 codrescence –70, whicOver the last few years, perfluorocarbon (PFC) nanodroplets have been developed into powerful probes for optical molecular imaging as well as image-guided treatments , 72. ForSemiconducting polymer nanoparticles (Pdots or SPNs) have recently generated tremendous interests as a novel class of contrast agents for biological imaging . They ex700/PA820) of 25, 7.3, and 2.7 times in solution, in cells, and in living mice (Pu et al. have developed a new class of near-infrared SPN probes for in vivo photoacoustic molecular imaging . SPN caning mice , respectIn this review, recent advances of both inorganic and organic nanoparticle probes for structural and functional photoacoustic tomography have been highlighted. The use of multifarious nanoprobes for various in vitro, ex vivo, and in vivo tests represents a surging trend in nanobiotechnology and nanomedicine. However, inorganic nanoparticles have the characteristic of being nonbiodegradable, which could accumulate within the animal body for a relatively long time. Importantly, the issue on the long-term toxicity is yet not resolved for preclinical and clinical investigations. In terms of UCNPs, the efficiency of the UC process would be much lower due to the solvent relaxation problem in aqueous conditions, which should significantly affect their potential applications for in vivo photoacoustic molecular tomography. Alternatively, organic nanoparticles including semiconducting polymers show good biocompatibility as demonstrated in different cellular assays –87. In t


The grant number is GR09/0021.In the Funding section, the second grant number from the funder Instituto de Salud Carlos III (Madrid, Spain;


Imaging techniques play a key role in the management of patients with colorectal cancer. The introduction of new advanced anatomical, functional, and molecular imaging techniques may improve the assessment of diagnosis, prognosis, planning therapy, and assessment of response to treatment of these patients. Functional and molecular imaging techniques in clinical practice may allow the assessment of tumour-specific characteristics and tumour heterogeneity. This paper will review recent developments in imaging technologies and the evolving roles for these techniques in colorectal cancer.Imaging techniques play a key role in the management of patients with colorectal cancer.• Advanced imaging techniques improve the evaluation of these patients.• Functional and molecular imaging allows assessment of tumour hallmarks and tumour heterogeneity.•  Colorectal cancer (CRC) represents one of the most commonly diagnosed cancers worldwide. It is the second most common cause of cancer death in the western world . A multiConventional imaging techniques have clear limitations for the evaluation of important tumour features. For example, 9-10 % of patients with computed tomography (CT)-indeterminate lung and/or liver lesions during radiological staging of CRC had definite metastases , 4. BesiConventional imaging techniques play a central role in CRC because they depict relationships of the tumour to surgical landmarks , the presence of important prognostic features, evaluate tumour response to treatment, and are useful for surveillance after therapy. In the case of rectal cancer (RC), magnetic resonance imaging (MRI) is the best imaging technique for evaluating main factors that affect treatment and prognosis, including tumour length, location from the anal verge, relationship to the peritoneal reflection, T-stage, depth of extramural tumour growth, lymph node (LNs) status, vascular and neural invasion, distance to the mesorectal resection margin, and invasion to adjacent structures , 9. BesiComputed tomographic colonography (CTC) involves the use of a CT scanner to produce 2- and 3-dimensional (3D) images of the entire colon and rectum obtained after air insufflation Fig. . CTC canb-values images due to the T2 shine-through effect can make the tumour segmentation on DWI not accurate.Tumour sizes and volumes have been proven to be an important prognostic indicator for a variety of tumours. However, these features were not found useful in the TNM staging system in CRC and in predicting the clinical outcome of patients, though published papers are sometimes contradictory –16. SimpHepatic resection has improved the survival of patients with metastatic CRC. Approximately 25 % of newly diagnosed patients with CRC have liver metastases at the time of diagnosis and another 25 % will develop liver metastases during the course of the disease. There are several key features to consider when planning hepatic resection, including the number of segments involved, the proximity of lesions to vascular and biliary structures, and the amount of remnant liver following resection. The size of the remnant liver affects procedural success and postoperative mortality and morbidity –22. ThisDual-energy computed tomography (DECT) is a new technique that allows for differentiation of materials and tissues based on CT density values derived from two synchronous CT acquisitions at different tube potentials in a single acquisition. Iodine uptake can be distinguished from other materials owing to its stronger photoelectric absorption at low tube voltages . DECT imMalignant tumours display a great spatial and temporal heterogeneity in their biological characteristics and behavior. However, much of the heterogeneity visible on imaging may represent noise. Texture analysis can reduce the effect of noise in images, while enhancing biologic heterogeneity. Texture analysis focuses on the distribution and relationships of grey-level values within images. Texture extracts basic components  from conventional images, creates a derived set of sub-images, and allows the quantification of different parameters, including entropy, kurtosis, and standard deviation of the pixel distribution histogram . In the CRC typically show characteristic tumour phenotypic alterations, which are manifestations of genetic changes and metabolic reprogramming, including sustained angiogenesis, limitless replication potential, and altered metabolic pathways (including an increased glycolytic capacity) . AnatomiAngiogenesis is a prognostic feature in CRC that has been correlated with important tumour characteristics such as grade and stage and with an increased incidence of metastases and local tumour recurrence. Functional imaging techniques may provide additional insights into the tumour microenvironment. Main imaging techniques for assessing tumour vascularization in the clinical field are DCE techniques based on MRI and CT. These techniques acquire a series of images through a region of interest before, during, and after the intravenous injection of a contrast media , 32. Diftrans) have demonstrated a prognostic value. Lim et al. reported that Ktrans values in rectal tumours at presentation in the downstaged group following CRTP were significantly higher than those in the non-downstaged group . Pr. Pr74]. 18F-3-deoxy-3-fluorothymidine (FLT) is a radiotracer, which allows the evaluation of cellular proliferation. Nonetheless, FLT-PET evidenced a limited value in CRC, demonstrating less sensitivity than 18F-FDG-PET for the detection of pathologic LNs or liver metastases in CRC [s in CRC , 76.Tumour proliferation needs nutrients, energy, and biosynthetic activity. This feature is responsible for the metabolic reprogramming associated with cancer. Imaging techniques allow assessment of the status of the altered metabolic pathways in CRC.2 concentration, a process named aerobic glycolysis. Proliferating tumour cells generally consume glucose at a high rate. This increased glucose uptake is the basis for clinical PET imaging in tumours. PET-CT is an established clinical technique for the management of CRC patients, which may have an impact on changing patient management strategies with its evolving role in diagnosis, radiation therapy planning, prediction of response, and therapy assessment [18F-FDG-PET data after preoperative CRT is predictive of OS in RC [18F-FDG-PET-CT in 98 % of patients [18F-FDG uptake is dependent on tumour grade and histological type, and PET shows a limited and a poor spatial resolution .A well-known energy metabolism alteration in tumour cells is an increased glycolytic capacity, even in the presence of a high Osessment –80. PET-sessment , 78. Nonsessment . Some pasessment , 81–83  MR spectroscopy (MRS) in CRC. Kim et al. demonstrated that RC mainly showed elevated choline (Cho) at 3.2 ppm and lipids (Lip) peaks at 1.3 ppm on MRS -MRI may provide a non-invasive means of assessing in-vivo tumour oxygenation based on endogenous deoxyhemoglobin as a contrast agent Fig. . HoweverNodal metastases are one of the most significant indicators of local recurrence and cancer-specific mortality in RC patients, and influence the determination of surgical and adjuvant treatments. CT, MRI, and endorectal ultrasound lacked sufficient accuracy to identify metastatic LNs with sensitivities and specificities in the 55–78 % range. Apart from this, histological data show that up to 45 % of nodal metastases in RC are ≤4 mm, which increases the difficulty of the accurate characterization of LNs. The development of ultrasmall iron oxide particle (USPIO) contrast agents opened the possibility of performing MR lymphography (MRL) . The conThe use of gadofosveset  can also significantly improve the diagnostic performance to discriminate between benign and metastatic LNs in RC. The intravenous administration of this contrast media results in a selective uptake of contrast in benign LNs, which causes an increase in signal on T1-weighted images, and enhances the chemical shift artfact around the border of the nodes .Combining the anatomical resolution of imaging with functional (such as DWI and DCE imaging) and molecular (PET) techniques may offer additional information about treatment of tumour microenvironments in different clinical scenarios, including staging, tumour characterization, prediction of response to treatment, and response evaluation –101. TheIn conclusion, advanced imaging techniques offer great opportunities in the evaluation of patients with CRC. The introduction of functional and molecular imaging techniques in clinical practice allows for the assessment of tumour hallmarks and tumour heterogeneity, which may change the management and therefore the prognosis of patients.


To evaluate whether early reductions in CT perfusion parameters predict response to pre-operative chemotherapy prior to surgery for gastroesophageal junction (GEJ) and gastric cancer.Twenty-eight patients with adenocarcinoma of the gastro-esophageal junction (GEJ) and stomach were included. Patients received three series of chemotherapy before surgery, each consisting of a 3-week cycle of intravenous epirubicin, cisplatin or oxaliplatin, concomitant with capecitabine peroral. The patients were evaluated with a CT perfusion scan prior to, after the first series of, and after three series of chemotherapy. The CT perfusion scans were performed using a 320-detector row scanner. Tumour volume and perfusion parameters  were computed on a dedicated workstation with a consensus between two radiologists. Response to chemotherapy was evaluated by two measures. Clinical response was defined as a tumour size reduction of more than 50%. Histological response was evaluated based on residual tumour cells in the surgical specimen using the standardized Mandard Score 1 to 5, in which values of 1 and 2 were classified as responders, and 3 to 5 were classified as nonresponders.A decrease in tumour permeability after one series of chemotherapy was positively correlated with clinical response after three series of chemotherapy. Significant changes in permeability and tumour volume were apparent after three series of chemotherapy in both clinical and histological responders. A cut-off value of more than 25% reduction in tumour permeability yielded a sensitivity of 69% and a specificity of 58% for predicting clinical response.Early decrease in permeability is correlated with the likelihood of clinical response to pre-operative chemotherapy in GEJ and gastric cancer. As a single diagnostic test, CT Perfusion only has moderate sensitivity and specificity in response assessment of pre-operative chemotherapy making it insufficient for clinical decision purposes. Computed Tomography (CT) perfusion can visualise changes in tumours vascular physiology, and could thereby potentially provide a biomarker for therapeutic response to chemotherapy In patients with locally advanced esophageal cancer, gastroesophageal junction (GEJ) cancer and gastric cancer perioperative chemotherapy improves overall and progression-free survival as confirmed by several randomised trials and meta-analyses Our hypothesis was that a reduction in perfusion parameters could predict treatment response of pre-operative chemotherapy. To address this hypothesis changes in CT perfusion parameters in GEJ and gastric cancer during preoperative chemotherapy were measured and analysed for correlation with both clinical and histological response.From October 2011 to January 2013 thirty consecutively admitted patients with biopsy confirmed adenocarcinoma at the gastro-esophageal junction or in the stomach were included in the study after being considered potentially resectable at a multidisciplinary tumour (MDT) conference and offered preoperative chemotherapy. Exclusion criteria were: allergy to contrast material, impaired renal function, and patient unfitness for chemotherapy. Of these thirty patients, one patient completed only the first scan and one patient was excluded from the study due to motion artefacts appearing in all three perfusion scans. The remaining 28 patients constituted the study group . Twenty-six patients underwent all three scans as described below. One patient was excluded from the third scan due to elevated serum creatinine levels, and one patient did not undergo the second scan due to hardware failure, leaving 27 patients for evaluation after one series of chemotherapy and 27 for presurgical responses. The diagnostic workup consisted of an upper endoscopy with biopsy, CT of the chest and abdomen combined with ultrasound of the neck and/or PET-CT for tumour staging, and after MDT conference a diagnostic laparoscopy to rule out peritoneal carcinomatosis. Patients with locally advanced tumour (T2– T4), with or without lymph node involvement (N0– N3) and no distant metastases (M0) were included. Methods and results are presented according to the REMARK recommendations for biomarker studies The research protocol was approved by the Committees on Biomedical Research for the Capital Region of Denmark (protocol number H-1-2010-132). All patients gave oral and written informed consent according to the Helsinki II Declaration.All CT perfusion examinations were performed using a 320-detector row CT scanner . The study protocol consisted of three perfusion scans: baseline , after the first cycle of chemotherapy  and - before surgery - after three cycles of chemotherapy . The last scan was performed at a median of 6 days before surgery (range 1–21 days).I.V.) as a gastrointestinal motility inhibitor. An abdominal strap was placed around the patient to reduce movement and to encourage shallow breathing during the examination.Each patient was instructed to fast 2-hours prior to the CT examination, and was given 500 mL of water just prior to the CT perfusion study to distend the stomach for better visualisation of the tumour. The anticholinergic, hyoscine butylbromide, was administered  was used as a test-bolus  to determine the initial delay from contrast administration to arrival in the aorta. The arrival of contrast was visually determined and the delay for the CT perfusion scan was set to assure the acquisition of 2 full scan volumes without contrast material in the dynamic sequence.The CT perfusion scan protocol consisted of 19 consecutive volumes divided into 3 phases: The first phase consisted of 11 volumes at 2 second intervals, a second phase of 6 volumes at 3 second intervals, and a third phase of 2 volumes at 5 second intervals. The first volume scan started between 7.5 and 13.5 seconds after contrast administration, and the total acquisition time ranged from 55 to 60 seconds. The contrast volume ranged from 30 to 40 mL, dependant on patient bodyweight  followed by a saline flush of 30 mL. The injection rate varied from 5 to 8 mL/s, so that the overall contrast injection time never exceeded 5 seconds. The image acquisition used the following parameters: 100 kV, 100 mA, 0.5 s/rotation time, fixed table position, and 0.5 mm reconstruction.Images were reconstructed using AIDR 3D . All datasets were corrected for motion using a non-rigid registration algorithm on the scan console (Toshiba). The volume containing the peak aortic enhancement was selected as a reference phase for motion correction. In two cases, the two late volumes were scanned at a different table position, and could therefore not be included in the motion correction.2 circular Region of Interest (ROI) in the centre of the abdominal aorta and a second ROI was placed in the tumour. Parametric perfusion maps were generated with the following perfusion parameters: Arterial flow (tissue perfusion measured in mL⋅min−1⋅100 g−1), blood volume (measured in mL⋅100 g−1) and permeability (measured as ktrans in mL⋅min−1⋅100 g−1). Based upon consensus between two radiologists, a free-hand ROI was drawn around the tumour-border on slices containing visible tumour and these circumscribed areas were utilized to define tumour volume using a sculpt-tool. Care was taken to exclude esophageal and gastric lumen and surrounding tissue. A ROI was also drawn on baseline scans in healthy appearing gastric tissue.The reconstructed, motion-corrected datasets were transferred to a stand-alone workstation  for post-processing. For data analysis, the input artery was selected by placing a 100 mmPatients were Scheduled for Three Cycles of Chemotherapy Preoperatively. after Completion of the Study, Patients Were Scheduled for Postoperative Chemotherapy, in Accordance with the MAGIC Study In patients with tumour at the gastro-esophageal junction an Ivor-Lewis Esophagectomy with extended D1+ lymphadenectomy was performed. In patients with a stomach tumour a complete gastrectomy was performed. One patient  was not resected due to local tumour growth invading the pancreas and was because of this classified as a clinical nonresponder without histological grading.Responses were evaluated both clinically and histologically. Investigators performing the evaluations were blind to patient outcome and results of the imaging studies. For the clinical response evaluation, the endoscopic imaging and tumour length, measured with a straight endoscope at the level of the incisors, before chemotherapy was compared with the macroscopic measurements of tumour in the surgical specimen – taking tumour shrinkage of 10% due to fixation into account Tumour perfusion parameters at baseline were compared with perfusion parameters from normal gastric tissue. Changes between tumour perfusion parameters at baseline and the second, and third scans, were calculated using the following formulas, respectively, where brackets represent a perfusion measure:All statistical tests were performed using SPSS . Perfusion parameters at baseline and at the third scan were compared between responders and nonresponders using Mann-Whitney U-test. Changes in tumour perfusion between first and second scans, and changes between first and third scans were compared between groups based on response type using univariate logistic regression. Comparisons of tumour tissue perfusion and normal gastric tissue perfusion was performed by using a Wilcoxon signed-rank test. A 2-sided p-value below 0.05 was considered statistically significant. A Receiver Operating Characteristic (ROC) analysis was made to determine the optimal diagnostic cut–off value for changes in perfusion parameters in regards of sensitivity and specificity for predicting clinical response.−1⋅100 g−1 versus 77.2 mL⋅min−1⋅100 g−1, p<0.01).Of the 28 patients available for clinical response evaluation, 13 were classified as clinical responders and 15 were classified as nonresponders. One patient was not resected due to local tumour growth invading the pancreas and was because of this classified as a clinical nonresponder. Based on histological examination, eight cases  were classified as responders and 19 cases  were classified as nonresponders. Using ROC analysis , a cut-oAfter three series of chemotherapy, the probability of responding was correlated with a decrease in tumour permeability (p = 0.03). A decrease in tumour volume was also positively correlated with response (p = 0.01). The absolute change in blood volume was positively correlated with response (p = 0.03), but not in percentage change (p = 0.08).The logistic regression analysis showed that the probability of a histological response increased with a reduction in permeability (p = 0.03) and in tumour volume (p = 0.03) between baseline scan and after third series of chemotherapy. Changes in arterial flow and blood volume were not significant . We observed no significant changes after the first series of chemotherapy in arterial flow (p = 0.39), blood volume (p = 0.42), permeability (p = 0.20), or tumour volume (p = 0.25). Comparison of perfusion parameters and tumour volume of the preoperative scan with histological response showed a significant difference in permeability between histological responders and nonresponders (p<0.05), as demonstrated by clinical response. Likewise, responding patients had significantly smaller tumours than non-responding patients (p<0.01). There were no statistically significant differences in blood volume (p = 0.31) and arterial flow (p = 0.43) .Within the first year after inclusion 8 out of the 27 resected patients (30%) had recurrence of disease. Four of these 8 were clinical responders after surgery  and 1 was a histological responder .trans) after one series of chemotherapy (three weeks) and treatment response after three series of chemotherapy measured by clinical response. This correlation appeared before significant changes in tumour volume were detectable in the scans. Patients with a histological response had a significantly lower tumour permeability compared to nonresponding patients. To our knowledge, this is the first longitudinal study using CT perfusion as a method to measure early response to pre-operative chemotherapy in GEJ and gastric cancer.The main finding of this study was that there was a positive correlation between a decrease in tumour permeability (ktrans), which is the rate constant for transfer between the intravascular and extravascular  spaces. The lack of standardisation of CT perfusion protocols, and implementation of kinetic models complicate direct comparison of values between studies.Perfusion is the delivery of blood through the vascular bed and can be estimated using different kinetic models. The basis of CT perfusion is the transport of a contrast material by blood flow through the intravascular space, and subsequently, the distribution of the contrast material between the intravascular and extravascular interstitial space The first adoptions of the CT perfusion technique were limited to single slice studies. With the use of a 320-detector row CT scanner it is possible to cover 16 cm in a single gantry rotation, thereby improving the temporal resolution compared to a helical scan The clinical response rate in our study was 46% (13/28), which is comparable to those of other response evaluation studies of esophageal and gastric cancer Other imaging modalities have been used for response assessment of preoperative chemotherapy for adenocarcinomas in the esophagus and at the gastroesophageal junction. Studies using Positron Emission Tomography (PET) with fludeoxyglucose (FDG) as tracer There are some limitations to our study. First, our study sample is small and the data presented should be interpreted with this in mind. Secondly, albeit GEJ and gastric cancer arise from the same epithelial cells, they are located at different sites in the gastrointestinal tract, with possible different outcome to treatment. There is consensus on treating the cancers in the same peri-operative setting, and the MAGIC trial trans) after one series of chemotherapy and the response after three series of chemotherapy, based on tumour size reduction. A cut-off value with a reduction in permeability of more than 25% gives a sensitivity of 69% and a specificity of 58% after one series of chemotherapy, which makes the diagnostic quality moderate and insufficient as a single discriminatory test. With regards to histological response, our study finds significantly lower permeability in responding tumours compared to non-responding after three series of chemotherapy, but not after the first series of chemotherapy. Histological response evaluation seems better than clinical response to estimate the risk of recurrence of disease.In conclusion, our study found a positive correlation between an early decrease in tumour permeability (kChecklist S1The REMARK checklist.Study details in accordance with reporting recommendations for tumor marker prognostic studies (REMARK).(DOC)Click here for additional data file.


Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga  C. elegans in wet soil13The motility of microorganisms and organelles through microstructured fluids plays an important role in varied biological processes such as fertilization and genetic transport123479ℓ, low swimming speeds U or both. In a Newtonian fluid with constant density ρ and viscosity μ, this implies that inertial effects are negligible and that the stresses felt by the swimmer are purely viscous and linear in the viscosity. Since the Stokes equations govern the fluid response to the moving body, the fluid kinematics is reversible. To therefore swim, organisms must execute non-reversible, asymmetric strokes in order to break free of the constraints imposed by the so-called “scallop theorem”λ may then become comparable to the viscous diffusion time of vorticity ρℓ2/μ. For microorganisms with cyclical swimming strokes, the elastic stresses may then persist between periods and eventually dominate over viscous effects.Many organisms move in the realm of low Reynolds number C. elegansC. reinhardtii) and “pushers” (such as E. coli). On the other hand, simulations of finite-sized movingtogether impact motility in a non-linear manner12The consequences of fluid elasticity on the details of swimming while clearly important, are not well-understood, and have therefore received growing attention4141617181920212218231617252616Chlamydomonas reinhardtii. With an ellipsoidal cell body  and enhance the alga's beating frequency and wave speed. Despite this enhancement, the alga's swimming speed is overall hindered (as much as 50%) by fluid elasticity due to the elastic stresses in the fluid.In this manuscript, we experimentally investigate the effects of fluid elasticity on the swimming behavior of the bi-flagella green alga, ell body  that is 6, Polysciences) to water. The polymer concentration in solution ranges from 5 to 80 ppm resulting in fluid relaxation times λ that range from 6 ms to 0.12 s, respectively  in M1 buffer solution. The Ficoll concentration in M1 buffer is varied from 5% to 20% by weight in order to produce fluids with a range of viscosities (1 cP to 10 cP). Viscoelastic fluids are prepared by adding small amounts of the flexible, high molecular weight polymer polyacrylamide  or viscoelastic (PAA) fluids. A small volume of this suspension is then stretched to form a thin film (thickness ≈ 20 μm) using a wire-frame device  and polymeric solutions (VE) of similar viscosity μ ≈ 6 mPa·s are presented in μ ≈ 2.6 mPa·s). The difference in shapes is striking and illustrates the effects of fluid elasticity on swimming. In the Newtonian case (i), the flagellum seems more mobile and significant changes in curvatures are attained over the whole cycle. In the viscoelastic case (ii), lateral displacements of almost a third of the flagellum close to the cell body (green) appear to be severely restricted (less mobile) or bundled together with most of the bending occurring over the remainder of the length. Furthermore, we observe localized bending at the distal tip in the initial stages of the power stroke. The differences in the shapes can be quantified by plotting the spatio-temporal evolution of the scaled flagellum curvature μ < 2.6 mPa·s), the distal tip gets closer to the cell body than for higher viscosity fluids (t (≈6 cycles) and dimensionless arc length s/ℓ, here denoted by 〈κ〉. We find that at μ ≈ 6 cP, the value of 〈κ〉 is −1.44 and −2.72 for the Newtonian and viscoelastic fluids, respectively.The beating pattern of y cycles . These ky fluids . We can μ ~ 2 mPa·s and lower, the frequency is roughly around 56 Hz. Increasing the viscosity further (μ > 2 mPa·s) results in a monotonically decreasing frequency. Intriguingly, we find that the decay is well captured by These changes in the spatio-temporal dynamics of the flagella translate to variations in beat frequency and swimming speed. We begin by investigating the effects of shear viscosity on the beat frequency  and on tU of the C. reinhardtii cell body decreases as the fluid viscosity increases for both Newtonian and polymeric solutions. For low viscosity values (μ < 2 mPa·s), the swimming speeds are very similar for the algae cells in Newtonian and polymeric liquids. As polymer concentration increases, however, we find that fluid elasticity consistently hinders self-propulsion compared to Newtonian fluid at comparable viscosity. For both Newtonian and polymeric fluids, we find the data consistent with the relationship U ~ μ−1, which suggests that the algae are operating at nearly constant thrust. Such a relationship has also been observed for free swimming C. reinhardtii in low viscosity Newtonian fluids (1 < μ < 2 mPa·s)The observed increase in beating frequency, however, does not translate into an increase in overall swimming speed. ωλ where ω is the mean frequency of a representative sample of cells and λ is the fluid relaxation time; we note that De = 0 for Newtonian fluids. The normalized beating frequency and algal swimming speed as a function of De are shown in 16The data shown in U+ and the mean speed during the recovery stroke U− for Newtonian and viscoelastic fluids. U+ and recovery U− stroke speeds decrease as μ increases, following the trend observed earlier for the Newtonian net swimming speed in U+ is similar to the Newtonian case, the recovery speed U− in viscoelastic fluids remains relatively unchanged, and in fact modestly increases with viscosity. This raises the possibility that fluid elasticity affects power and recovery strokes very differently - a view supported by plots of the normalized power and recovery stroke speeds shown in The net frequency and swimming speed while central to the alga's overall motility, do not distinguish between the highly asymmetric power and recovery strokes. Therefore, we next calculate the mean speed during the power stroke s ≈ 0 throughout the cycle, while for the Newtonian case , including mammalian spermatozoa progressing in human mucus (of different viscosities)2418μ = 5.7 cP). Indeed, recent theoretical analysisC. elegansIn this work, we focus on the algae μm using an adjustable wire-frame deviceω were on the order of 30–60 Hz and the Deborah number 0 ≤ De < 7 with Re ~ 10−3. Under these conditions, the dimensionless Elasticity number A small amount of motile alga was added to the solutions thus prepared and the suspension was further stabilized by adding the surfactant Tween-20. A small volume of this suspension was stretched into a thin film of thickness ≈20 C. reinhardtii centroid position. Raw images of the algae swimming first undergo image processing such as contrast enhancement for stronger signal on the body and band pass filtering for the removal of the high-frequency noise in the image. The centroid and the orientation of the cell body are then obtained using a non-linear least square method akin to template matching. The magnitude of the instantaneous swimming speed (or signed speed) is the ratio of the magnitude of the displacement vector over the time step. The sign of the instantaneous speed, i.e. from forward to backward or vice versa, is determined by inspecting the angle between the velocity and the cell body orientation. The net speed is the averaged speed averaged over multiple beating cycles . The shapes relative to the center of the cell body obtained from multiple sequential periods are then mapped to points along a cyclical trajectory of constant period  - the fiB.Q., J.Y., J.P.G. and P.E.A. designed experiments; J.Y. and B.Q. performed experiments; B.Q., A.G. and J.Y. analyzed experimental data; A.G., B.Q. and P.E.A. developed theories to interpret analysis; A.G., B.Q. and P.E.A. wrote the manuscript; all authors discussed and interpreted results; J.P.G. and P.E.A. conceived and supervised the project.SI - Text and FiguresSIRegular - SI Movie 1SIWobble - SI Movie 2SIWave - SI Movie 3


Varicella-zoster virus (VZV) is a highly neurotropic virus that can cause infections in both the peripheral nervous system and the central nervous system. Several studies of VZV reactivation in the peripheral nervous system (herpes zoster) have been published, while exceedingly few investigations have been carried out in a human brain. Notably, there is no animal model for VZV infection of the central nervous system. In this report, we characterized the cellular environment in the temporal lobe of a human subject who recovered from focal VZV encephalitis. The approach included not only VZV DNA/RNA analyses but also a delineation of infected cell types . The average VZV genome copy number per cell was 5. Several VZV regulatory and structural gene transcripts and products were detected. When colocalization studies were performed to determine which cell types harbored the viral proteins, the majority of infected cells were astrocytes, including aggregates of astrocytes. Evidence of syncytium formation within the aggregates included the continuity of cytoplasm positive for the VZV glycoprotein H (gH) fusion-complex protein within a cellular profile with as many as 80 distinct nuclei. As with other causes of brain injury, these results suggested that astrocytes likely formed a defensive perimeter around foci of VZV infection (astrogliosis). Because of the rarity of brain samples from living humans with VZV encephalitis, we compared our VZV results with those found in a rat encephalitis model following infection with the closely related pseudorabies virus and observed similar perimeters of gliosis.IMPORTANCE Investigations of VZV-infected human brain from living immunocompetent human subjects are exceedingly rare. Therefore, much of our knowledge of VZV neuropathogenesis is gained from studies of VZV-infected brains obtained at autopsy from immunocompromised patients. These are not optimal samples with which to investigate a response by a human host to VZV infection. In this report, we examined both flash-frozen and paraffin-embedded formalin-fixed brain tissue of an otherwise healthy young male with focal VZV encephalitis, most likely acquired from VZV reactivation in the trigeminal ganglion. Of note, the cellular response to VZV infection mimicked the response to other causes of trauma to the brain, namely, an ingress of astrocytes and astrogliosis around an infectious focus. Many of the astrocytes themselves were infected; astrocytes aggregated in clusters. We postulate that astrogliosis represents a successful defense mechanism by an immunocompetent human host to eliminate VZV reactivation within neurons. Varicella-zoster virus (VZV) is an alphaherpesvirus that can cause infections in both the peripheral nervous system (PNS) and the central nervous system (CNS). The neurotropism of VZV is manifested most commonly in the PNS as herpes zoster (shingles) . Herpes 3–Upon reactivation from the trigeminal ganglion, herpes zoster ophthalmicus is the prototypical expression of trigeminal zoster. In addition to innervation of the skin of the upper face, sensory afferent fibers originating in the V1 and V2 branches of trigeminal ganglion travel into the meninges of the frontal and temporal lobes, respectively , 7. FurtIn contrast to peripheral herpes zoster, samples of brain tissue from a human who recovers from a VZV infection of the CNS are exceedingly rare. We previously published a brief case report of focal VZV encephalitis . The lesFollowing an extensive literature search regarding alphaherpesvirus encephalitis, it was apparent that almost no neuropathology data were available about the cellular response to VZV encephalitis in a human subject who overcame his infection, as opposed to VZV-infected brain samples collected postmortem –11. Post–Griffonia (Bandeiraea) simplicifolia plant lectin I, isolectin B4 (IB4) (Invitrogen 121412), against microglia. For the reagents not conjugated with a fluoroprobe, we purchased the following secondary reagents: Alexa 488, 546, and 633 fluoroprobes conjugated to a goat anti-rabbit IgG, goat anti-mouse IgG, or goat anti-human IgG (Invitrogen).The anti-VZV reagents included the following monoclonal antibodies (MAbs): 3B3 (anti-glycoprotein E [anti-gE]), 233 (anti-gC), 206 (anti-gH), and human anti-gH. All murine MAbs have been characterized –15; like–The brain biopsy specimen had been flash-frozen at −70C. For confocal microscopy, several 10-μm frozen sections were cut in a cryostat and adhered to glass slides. The sections were incubated with antibody probes by the described methods and then viewed with a Zeiss LSM710 spectral confocal microscope, using 10×, 20×, 40×, and 63× high-numerical-aperture oil immersion objective lenses , 18. Ima4 plus 1.5% KFe(CN)6 in PBS for 2 h; washed twice with water, and then further stained with 2% uranyl acetate in water for 30 min. The tissue was then dehydrated in a graded series of ethanol solutions: 25%, 50%, and 75% for 15 min each followed by 100% ethanol twice for 30 min each, 50% ethanol–Epon (Eponate 12) for 2 h, 100% Epon for 2 h, and 100% Epon for 18 h. The tissue was then polymerized with 100% Epon at 70°C for 18 h. Microtomy yielded thin sections of the embedded brain tissue that were then placed on TEM grids, further stained with 4% uranyl acetate in water and then 1% lead citrate with water, washing after each stain. The grids were then air dried and imaged using a JEOL 1230 transmission electron microscope fitted with a Gatan camera.For electron microscopy, the frozen brain sample was thawed to approximately −10°C, and then a small piece (2 by 2 by 2 mm) was excised. That piece was further subdivided and the sections placed in BEEM capsules containing 2% glutaraldehyde in phosphate-buffered saline (PBS) at 4°C. The samples were then prepared for transmission electron microscopy (TEM) using previously described methodology . Brieflyad libitum. On the day of the experiment, the animals were anesthetized with isoflurane, and their heads were secured in a stereotaxic frame. Following shaving of the head and sterilization of the scalp with Betadine, an incision was made to expose the dorsal surface of the skull. A beveled 1-μl Hamilton syringe loaded with PRV strain RV-152 was lowered into the interpositus nucleus through a craniotomy drilled at coordinates obtained from the rat brain atlas of Swanson  laboratory for 2 days prior to the onset of the experiment. The temperature (22 to 25°C) and light-dark cycle  were standardized in the colony room, and food and water were available  Swanson . Virus w Swanson . AlternaGuide for the Care and Use of Laboratory Animals  was 8 cycles later for the brain tissue than the positive control, indicating 28 fewer cells. Assuming there are 1 million cells in the positive control, then the small piece of brain tissue consisted of approximately 4,000 cells. In general, the level of VZV transcripts in the brain tissue was consistently less than that of the positive control by 24 to 26, a result suggesting that a productive infection was occurring in far fewer than 10% of the cells within the brain tissue.In an earlier report, we had only amplified one VZV gene (ORF62) from brain tissue . To bettAfter collecting the genomic data, we postulated that neurons would be the cell in which infection would be most easily detected. Therefore, we selected an established antibody probe for neurons and two anti-VZV monoclonal antibody probes. Based on the fact that VZV gE (ORF68) is an abundant gene product during VZV infection, we chose a well-characterized anti-gE MAb, 3B3, with high-affinity binding for a linear epitope that is known not to cross-react with uninfected brain tissue , 23. WheGriffonia plant isolectin B4, which adheres to the cell wall of microglia. As seen in In order to investigate whether virus was present in microglia, we performed a similar confocal microscopy experiment after labeling cells with the Immunodetection of glial fibrillary acidic protein (GFAP) is the standard method for identifying intermediate filaments in activated astrocytes . BecauseTo further assess the aggregates of infected cells, z-stacks containing 40 slices were converted by Imaris software into 3D animations; the 3D images confirmed that extensive VZV gH-positive antigen was found within GFAP-positive cells. A 2D frame from one of the animations shows a syncytium of astrocytes containing about 80 nuclei . BecauseIn order to further clarify the relationship of GPAP immunolabeling to these aggregates, we probed a larger section of the flash-frozen brain tissue with antibodies to VZV gH and the astrocytic marker GFAP and visualized at low magnification . A wide-To determine whether VZV can complete its replication cycle in astrocytes, we next investigated whether the true late (gamma-2) gC protein (ORF14) was detectable . As a poTo gain further insight into the role and extent of reactive gliosis in VZV-induced neuropathology, we examined a portion of the excised temporal lobe tissue using transmission electron microscopy (TEM). Observers at both the University of Iowa and the University of Pittsburgh independently examined ultrathin sections of the tissue and identified the cell types . SystemaExamination of the interface between degenerating neurons and the barrier astrocytes revealed clear evidence of phagocytic activity . AstrocyBecause the reactive astrogliosis observed in the samples of VZV-infected human brain tissue exhibited features similar to those previously documented in the rat CNS following PRV infection , we condA central finding of this report is that astrocytes are an important component of the CNS response to VZV neurotropism in humans who recover from their infection. The role of astrocytes during the CNS stress response is a subject of increasing investigation , 34. AstAstrocytes in the human cortex are classified into 4 subgroups: (i) interlaminar, (ii) protoplasmic, (iii) varicose projection, and (iv) fibrous . The firGliosis is the term that describes a proliferation of glial cells in the CNS as a response to injury . GliosisVaricellovirus genus as VZV; neither VZV nor PRV harbors the herpes simplex virus 1 (HSV-1) neurovirulence protein  infection in the CNS , 32. PRV protein . In an eWith regard to VZV infection in the brain, another question is why the antiviral property of astrocytes was not previously recognized. One explanation is that VZV meningoencephalitis and vasculopathy can occur in the absence of skin rash and would be difficult to diagnose . A seconFinally, the case in this report does not represent an example of VZV vasculopathy as a cause of the CNS neuropathology , 45. How