Astragalus Mongholicus Regulate the Toll-Like-Receptor 4 Meditated Signal Transduction of Dendritic Cells to Restrain Stomach Cancer Cells

Background

According to the traditional view, we depend on three methods to treat tumors; surgery, chemotherapy and radiotherapy. However, these methods have its own limitations in application. Traditional Chinese Medicine (TCM) is one of the oldest healing systems. Astragalus mongholicus (AMs) that is the common herbal medicine, the biggest part of TCM, have been proved to be effective in treating cancers from lots of clinical cases. However, we have not fully understood the anti-tumor mechanism of AMs, and this has lead to some doubt for some Western-Medicine scholars and restricts its wide use. The main objective of this research is to discuss the effect and mechanism of AMs to human stomach cancer.

Materials and Methods

To observe the effect and mechanism of tumor treatment by AMs, we have done the research from three major aspects, the influence of DCs, the inhibition of tumor in vitro as well as the animal studies in vivo after treatment. First, we culture the mouse dendritic cells (DCs) from bone marrow of mouse hind legs according to the method using Interleukin-4(IL-4) and Granulocyte-macrophage colony stimulating factor (GM-CSF), which refer to the way established by Inaba (Inaba K, 1992). And then we investigate the growth-rate of the DCs co-cultured with AMs injection. We analyze the expression of the Toll-like-receptor 4 (TLR4), with SYBR-Green I Real-time PCR and the I-kappa-B-alpha (IκB-α) with Western-Blot, the main regulatory protein to control nuclear factor NFκB-p65 nuclear translocation. Second, we choose the human gastric cancer cell lines MKN 45 as the target cell, which was co-cultured with DCs, T cells from spleen of mouse and AMs injection, and use MTT assay to judge the amount of cell lines and Immnunoflurescene to analyze the expression of anti-active caspase 3 pAb anti-PARP P85 fragment pAb, the mark of apoptosis of cells. Third, we have conducted the animal studies beside the basic experiment in vitro. The nude mouse developed stomach cancer, due to intra-preritoneal injection with MKN45 have been divided into two groups: the treatment group challenged with AMs injection and the control group with saline injection. We took the average of the diameter of each group as the y axis and the days after administered with AMs as x axis. After 40 days, all animals were killed by detruncation, and the tumor were removed and measured. We compare the diameter (<40 days) and weight (>40 days) of the tumor as well as the survival days between different groups to investigate the effect of inhibition of cancer.

Results

All results show that AMs is effective in treating human stomach cancer and the mechanism might be regulated by TLR4 mediated signal transduction of DCs. The results are briefly introduced as follows: First, we succeed in culturing the DCs induced by IL-4 and GM-CSF and find the positive rate of CD11c expression, the mark of DCs, is beyond 90% (Fig-1). We detect AMs can precipitate DCs maturation by upregulating TLR4 in SYBR-Green I Real-time PCR (Fig-2) and suppressing I.B-aby Western-Blot (Fig-3). Second, after the MKN45 co-cultured with DCs, T cells and AMs injection, the result show that AMs can great reduce the amount of cell lines by MTT assay (Fig-4) and induce apoptosis with Immunofluorescence (Fig-5). Finally, we have conducted animal studies beside the experiment in vitro, and the result in vivo show that AMs can delay tumor development from the diameter and weight of the tumor (Fig-6, Fig-7), prolong life-span and improve life-quality.Open in a separate windowFigure 1the morphology and phenotypic identification of DCs.

1. The form of DCs observed by microscope with field 20*.
2. The isotype antibody control using FCM.
3. The positive rate of CD11c expression.

Open in a separate windowFigure 2the melting curve and the chart of TLR4 expressiona) the melting curve of beta-actin; b)the melting curve of TLR4;c)the TLR4 expression of DCs stimulated with AM at different dose. There is significant statistic difference between the 60ng/mL and 80ng/mL group and other group (P<0.05 by rank test)Open in a separate windowFigure 3the IκB-α expression of DCs with different dose of AMsL0: 0ng/mL; L1:20ng/mL; L2:40ng/mL; L3:60ng/mL; L4:80ng/mLOpen in a separate windowFigure 4MTT assay to analyse the viability and proliferation of the two cell lines (P<0.05 between the group with the dose of 60ng/mL and 80ng/mL and other group). the horizontal axis is the group treated with AM and saline at different dose, the vertical axis is the cell number.Open in a separate windowFigure 5the anti-active caspase-3 pAb (a) and anti-PARP P85 fragment pAb (b) actived by immunofluorescence.The cell mix were treated with 100uL anti-active caspase-3 pAb at a 1:250 dilution and anti-PARP P85 fragment pAb at a 1:100 dilution, and the secondary Ab was donkey anti-rabbit Cy®3 conjugate diluted 1:500 in PBS (Jackson Cat#711-165-152). From the photo, we find that anti-active caspase-3 pAb and anti-PARP P86 fragment pAb can express which is very important to indicate cell-apoptosis.Open in a separate windowFigure 6The difference of tumor between treatment group and control group.

Conclusion

Ams Can play a great role in treating human stomach cancers as a good Chinese herbal medicine by precipitating DCs maturation, which is probably due to its effects by regulating the TLR4 mediated signal transduction.     

Transient Complete Heart Block Following Femoral Arterial Sheath Removal: An Extreme Case of Vasovagal Reflex Syndrome

How to cite this article: Kodliwadmath A, Walia R, Ola R, Sharma P. Transient Complete Heart Block Following Femoral Arterial Sheath Removal: An Extreme Case of Vasovagal Reflex Syndrome. Indian J Crit Care Med 2021;25(7):825–827.

Dear Sir,We report the case of a 65-year-old gentleman, nonhypertensive, nondiabetic, chronic smoker who presented to us with typical anginal discomfort for the past 2 weeks. Baseline 12-lead surface electrocardiogram (ECG) showed sinus rhythm with ST depression in V4 to V6; echocardiography showed no regional wall motion abnormality with a left ventricular ejection fraction of 60%; and troponin I was 0.02 µg/L (normal <0.12 µg/L). He was diagnosed as suffering from acute coronary syndrome—unstable angina—and started on medical management including dual antiplatelets, statins, anticoagulants, and antianginals. Because of the feeble radial pulses, he was taken up for coronary angiography from the right femoral route, which showed a right dominant circulation and a mid-left anterior descending (LAD) coronary artery having discrete 90% stenosis, while the other coronary arteries were normal. The patient underwent successful percutaneous coronary intervention (PCI) to mid-LAD using one sirolimus-eluting stent of size 3 × 16 mm. He was shifted to the coronary care unit for post-PCI management. After 6 hours of the PCI and when the activated clotting time was 120 seconds, the femoral arterial sheath was removed under all aseptic precautions by the manual compression method under adequate local anesthesia—10 mL of 1% lignocaine. Three minutes after sheath removal, the patient reported increasing uneasiness, nausea, yawning, and anxiety. His heart rate decreased from 86 beats per minute (bpm) to 49 bpm, blood pressure (BP) fell from 136/74 to 84/39 mm Hg, and the bedside monitor showed atrioventricular dissociation with complete heart block (CHB) with a ventricular rate of around 49 bpm, and narrow QRS complexes with junctional escape rhythm suggestive of intranodal block (Fig. 1). No femoral or jugular venous access was available for emergency temporary pacemaker insertion. In view of the clinical scenario suggestive of vasovagal reflex syndrome (VVRS), he was rushed with 500 mL of intravenous normal saline (0.9% NaCl) and 0.6 mg of atropine injection. Immediately after atropine injection, his heart rate improved to 110 bpm and BP improved to 110/72 mm Hg and the CHB reverted to sinus rhythm, even before it could be documented on a 12-lead ECG. ECG taken after achieving hemostasis showed sinus tachycardia and ST depression in V4 to V5 (Fig. 2). The rest course of the hospital stay was uneventful, and the patient was discharged with stable vitals on guideline-directed medical therapy.Open in a separate windowFigs 1A to CSeries of bedside patient monitor images showing bradycardia with a heart rate of 49 – 50 bpm, hypotension with a blood pressure of 84/39 mm Hg, normal oxygen saturation of 99 – 100%; and lead I electrocardiogram strip showing regular P-P intervals, narrow QRS complexes with regular R-R intervals with atrioventricular dissociation suggestive of intranodal CHB (third-degree atrioventricular block) (*indicates the P waves)Open in a separate windowFig. 2Twelve-lead surface electrocardiogram done post-atropine, after hemostasis after sheath removal was achieved, showed sinus tachycardia with a heart rate of 125 bpm and ST depressions in V4 to V5VVRS is a common complication following sheath removal, characterized by hypotension, sinus bradycardia, and rarely by sinus arrest.^1,2 Clinical presentation includes light-headedness, yawning, nausea, vomiting, diaphoresis, anxiety, bradycardia, and hypotension.³ Though sinus arrest for 4 to 10 seconds is known after sheath removal,² CHB following sheath removal has not been described in the literature to the best of our knowledge. The treatment of VVRS includes intravenous fluids and atropine injection.⁴ Our case shows that VVRS in the extreme case can lead to CHB with narrow QRS and junctional escape rhythm at rates of 40–60 bpm indicating an intranodal block.⁵ Thus, along with monitoring of pulse and BP, continuous cardiac rhythm monitoring during sheath removal is of utmost importance as atrioventricular conduction disturbances including CHB can rarely occur, with a quick response to atropine.     

Mucin as possible cause of early adhesional intestinal obstruction

Objectives

To report the case of a 24 year old female undergraduate who presented with bowel obstruction, three weeks following the excision of a mucinous ovarian cyst.

Patient and methods

The records of the patients past and recent medical history laboratory and imaging studies were reviewed.

Results

Clinical findings of a distended and a plain abdominal radiogram showing distended loops of bowel(Figure 2) were in keeping with acute bowel obstruction. This was confirmed by the intraoperative finding of fibrous encasement of small bowel. This was excised ,and 12months thereafter, patient has remain in good health.Open in a separate windowFigure 2Preoperative A-P abdominal X-ray in erect position showing small distension

Conclusion

Early and absolute adhesional bowel obstruction from abdominal surgery is failing conservative management rate. We attributed this to the ruptured mucinous cyst in our earlier operation. We therefore suggest that should a rupture cyst of this type occurs during a surgical procedure, any of the preventive methods discussed should be tried as a prophylactic measure.     

Symptoms of degeneration of the pyramidal tracts in conventional magnetic resonance imaging and diffusion tensor imaging in a young woman with primary lateral sclerosis

Primary lateral sclerosis (PLS) is one of the forms of motor neuron disease (MND), affecting only upper motor neurons. The diagnosis of PLS should be made on different diagnostic criteria, for example, Pringle or Gordon, but it is usually a diagnosis of exclusion. There are no characteristic findings in standard laboratory and electrophysiological assessment. We present details of a 31-year-old woman who had suffered from progressive paraparesis with right-side predominance. Conventional MRI of brain and spinal cord and diffusion tensor imaging (DTI) studies showed Hyperintense lesions in the upper part of the cervical spinal cord at the level C1 in lateral funicules, in the medulla oblongata at the pyramidal decussation and in the midbrain. Brain DTI revealed changes along the corticospinal tracts on fractional anisotropy (FA) maps. MRI of the thoracic spinal cord showed in T2-weighted images hyperintensive regions in the course of the lateral corticospinal tracts. This aided in PLS recognition.KEY WORDS: Diffusion tensor imaging, MRI, primary lateral sclerosisPrimary lateral sclerosis (PLS) is one of the forms of motor neuron disease (MND), affecting only upper motor neurons. PLS diagnosis should be based on different diagnostic criteria, for example, Pringle or Gordon, but it is usually a diagnosis of exclusion. There are no characteristic findings in standard laboratory and electrophysiological assessment.[1]A 31-year-old woman, after bone-marrow transplantation from an unrelated donor because of the paroxysmal nocturnal hemoglobinuria, was admitted thrice to our Neurological Department. She had suffered from progressive paraparesis with right-side predominance since August 2009 and her family history was negative.Her first hospitalization revealed enhanced straight reflexes in the upper limbs with right-side predominance, paraparesis of the lower limbs was equal to 3 points on the Lovett scale on the right side and 4 points on the left, deep reflexes were enhanced bilaterally. Babinski sign was positive on both sides; without sensory and cerebellar disturbances, bulbar and pseudobulbar symptoms, or bladder impairment. We noted an increase in the paraparesis between November 2009 and June 2010.Blood biochemistry, morphology, B12, level, cerebrospinal fluid with peptide immunoelectrophoresis analysis and IgG index were normal. Lues and intoxication of heavy metals were excluded. Borrelia burgdorferi, Herpes, ECHO, Coxackie, HTLV1 viruses antigens were negative.The latencies of the visual evoked potentials were slightly prolonged, auditory brain stem evoked potentials, somatosensory evoked potentials, EEG, electroneurography, electromyography, pain, temperature and vibration sensation thresholds were normal. EMG was normal during the whole time of the observation.Conventional brain MRI and spinal cord as well as diffusion tensor imaging (DTI) studies were performed with a 1.5T Signa Hdx MR scanner; we used a high resolution 16-channel HNS coil, brain DTI was performed with single-shot spin-echo-type echo-planar imaging. Brain MRI revealed the presence of areas of increased signal in T2-weighted images [Figure 1] and FLAIR sequence along the pyramidal tracts. Hyperintense lesions were visible in the upper part of the cervical spinal cord at the level C1 in lateral funicules [Figure 1a], in the medulla oblongata at the pyramidal decussation [Figure 1b] and in the midbrain — in the ventral part of the left cerebral peduncle [Figure ​[Figure1c1c and ​andd].d]. Hyperintense lesions in the left cerebral peduncle were also clearly visible in diffusion-weighted images (DWI) [Figure ​[Figure1e1e and ​andf].f]. Brain DTI also revealed changes along the corticospinal tracts on fractional anisotropy (FA) maps. Focal decrease of the FA parameter was visible at the C1 spinal cord level in the lateral funicule region [Figure 2a], at the pyramidal decussation [Figure 2b], with changes more strongly marked on the left side, and in the ventral left cerebral peduncle [Figure ​[Figure2c2c and ​andd].d]. MRI of the thoracic spinal cord showed in T2-weigted images hyperintensive regions in the course of the lateral corticospinal tracts [Figure ​[Figure3a3a and ​andb].b]. The signal of the lateral pyramidal tracts was also increased, but to a lesser extent in the T1-weighted images [Figure ​[Figure3c3c and ​andd].d]. There were no atrophies of the spinal cord.Open in a separate windowFigure 1Brain MRI, axial and coronal T2-weighted images showing increased signal intensity (arrows) along the corticospinal (pyramidal) tracts in lateral funicules at the C1 level of the spinal cord (a) at the level of medullary pyramids (b) and in the left cerebral peduncle (c and d). On diffusion-weighted images (DWI) (e and f) hyperintense lesions are clearly visible in the left cerebral peduncle (arrows)Open in a separate windowFigure 2Diffusion tensor imaging (DTI), fractional anisotropy (FA) maps reveal focal decrease in the FA values in the course of pyramidal tracts (arrows). As in structural MRI changes are visible in lateral funicules of the spinal cord at the level of C1 (a), in the pyramidal decussation (b) and in the left cerebral peduncle (c and d)Open in a separate windowFigure 3MRI, axial T2-weighted images show hyperintensities along the lateral pyramidal tracts (lateral funicules) at the level of the upper (a) and lower (b) thoracic spinal cord. Increased signal intensity of the lateral funicules is also visible on T1-weighted images without contrast medium (c and d)PLS recognition is based on clinical and EMG examination after the exclusion of other diseases, and according to either Pringle''s or Gordon''s criteria.[1] In our patient conventional brain MRI revealed hyperintense lesions in T2-weighted images along the pyramidal tracts. Similar changes were found in earlier neuroimaging and neuropathological studies in patients with MND.[2,3] Hyperintensity probably reflects the level of gliosis and demyelination.[3] We did not observe any changes in the cortex of the precentral gyrus and other cortical regions as other authors.[2,3,4] Focal decrease in the FA value on brain DTI maps found in the course of corticospinal tracts, hyperintensity in T2-weighted images in the lateral corticospinal tracts of the thoracic spinal cord indicated axonal degeneration of the pyramidal tracts in our patient.[3,4,5] Wang et al.[3] indicated the meaning of changes in posterior limb internal capsule (PLIC) in the diagnosis of PLS. In our case, the DTI showed areas of decreased FA value in PLIC, cerebral peduncles as well as in the spinal cord, mostly the C1 segment. To the best of our knowledge there have been few studies on MRI changes in the spinal cord in patients with different types of MND. The asymmetry of pyramidal tract lesions closely corresponded to the clinical findings (asymmetry of paresis). According to Agosta et al.[6] the DTI could be useful in differential diagnosis between PLS and ALS patients with the highest accuracy of corpus callosum FA values, but they did not perform MRI of the spinal cord.The authors emphasize the importance of neuroimaging in PLS recognition. A good quality, high-resolution MRI is able to visualize changes in pyramidal tracts at the level of the spinal cord and DTI study at the level of the brain in PLS patients. This suggests the utility of spinal cord abnormality disclosure as an analytical tool for PLIC changes in MND patients.     

B cell contribution to immunometabolic dysfunction and impaired immune responses in obesity

Open in a separate windowGraphical Abstract     

Multiple uterine perforations during manual vacuum aspiration: the need to increase the clinical awareness of attending healthcare professionals

BackgroundThe risk of uterine perforation during manual vacuum aspiration (MVA) is reduced by using Karman cannula (which has a rounded tip) during the procedure.MethodsA 35-year-old multigravida at 13 gestational weeks presented with vaginal bleeding of a day duration and ultrasound evidence of retained products of conception suggestive of incomplete miscarriage. The patient was rhesus D positive and stable. She had MVA which was performed using Karman cannula, and developed severe vaginal bleeding. The differential diagnoses were incomplete uterine evacuation and uterine perforation.ResultsDuring a laparotomy in Lloyd-Davies position, haemoperitoneum and six uterine perforations on the anterior and fundal parts, each approximately 5 mm in length (Figure 1), were found. The perforations were repaired and a check uterine curettage under oxytocic cover showed an empty uterus. The abdominal cavity was washed and closed. She was transfused three units of red blood cell concentrate and had a normal six weeks follow-up.Open in a separate windowFigure 1Uterine perforations (arrows) with Karman cannula during manual vacuum aspiration.ConclusionWhen an instrument inserted into the uterus is pushed beyond the estimated depth of the uterus, a perforation must be suspected and the condition may be managed conservatively. A surgical procedure complicated by surgeon''s loss of perception (in this case tactile) of tissues'' anatomy is hazardous.     

The first five years of single-cell cancer genomics and beyond

Single-cell sequencing (SCS) is a powerful new tool for investigating evolution and diversity in cancer and understanding the role of rare cells in tumor progression. These methods have begun to unravel key questions in cancer biology that have been difficult to address with bulk tumor measurements. Over the past five years, there has been extraordinary progress in technological developments and research applications, but these efforts represent only the tip of the iceberg. In the coming years, SCS will greatly improve our understanding of invasion, metastasis, and therapy resistance during cancer progression. These tools will also have direct translational applications in the clinic, in areas such as early detection, noninvasive monitoring, and guiding targeted therapy. In this perspective, I discuss the progress that has been made and the myriad of unexplored applications that still lie ahead in cancer research and medicine.A single cell is the ultimate denomination of a multicellular organism. The human body is composed of approximately 37.2 trillion cells that live harmoniously among their neighbors (Bianconi et al. 2013). However, in cancer, a single cell can lead to the downfall of an entire organism. As a single cell begins its journey to evolve into a malignant mass of tumor cells, the lineages diverge and form distinct subpopulations resulting in intratumor heterogeneity. Clonal diversity is a salient feature of many human tumors (Navin et al. 2010; Gerlinger et al. 2012; Shah et al. 2012) and provides fuel for evolution to select upon. A tumor is analogous to an ecosystem, and many principles from ecology and population genetics can help us understand how tumor cell populations respond to selective pressures (Merlo et al. 2006; Greaves and Maley 2012). Clonal diversity is likely to play an important role in invasion, metastasis, and the evolution of resistance to therapy (Navin 2014a). However, to date, our understanding of intratumor heterogeneity in the context of tumorigenesis has been poor—at best. The central problem inhibiting these studies is technical: Most genomic methods require micrograms of input material and thus are limited to reporting an average signal from a complex population of cells. To address this problem, single-cell sequencing (SCS) methods were developed and have revolutionized our understanding of cancer evolution and diversity. Additionally, these methods can provide insight into the role of rare cells to understand their role in tumor progression. Over the past five years, there has been extraordinary progress in the development and application of single-cell DNA and RNA sequencing methods for cancer research (Fig. 1). However, many technical challenges and unexplored applications still lie ahead and serve as the basis of this perspective article (cf. Fig. 2).Open in a separate windowFigure 1.Timeline of single-cell sequencing milestones in cancer research. This timeline depicts the major discoveries that have occurred in the cancer field of single-cell sequencing and related technology developments.Open in a separate windowFigure 2.Applications of single-cell sequencing in cancer research. This figure displays the myriad of applications that single-cell sequencing methods have in cancer research: (A) resolving intratumor heterogeneity; (B) investigating clonal evolution in primary tumors; (C) studying invasion in early stage cancers; (D) tracing metastatic dissemination; (E) genomic profiling of circulating tumor cells; (F) investigating mutation rates and mutator phenotypes; (G) understanding resistance evolution to therapy; (H) understanding cancer stem cells and cell hierarchies; and (I) studying cell plasticity and epithelial-to-mesenchymal transition.     

Discovering the 60 years old secret: identification of the World War II mass grave victims from the island of Daksa near Dubrovnik, Croatia

Aim

To describe the organization, field work, forensic anthropological examination, and DNA analysis conducted to identify the victims from a World War II mass grave found on the Dalmatian island of Daksa near Dubrovnik (Croatia) in 2009.

Methods

Excavation of the site was performed according to standard archeological procedures. Basic anthropological examination was made to determine the minimum number of victims, sex, age at death, and height. The bones with pathological and traumatic changes were identified. DNA was extracted from powdered bones and relatives’ blood samples. Y-chromosome and autosomal short tandem repeats (STR) were used to establish the relationship of the remains with the putative family members.

Results

The remains were found to belong to at least 53 distinctive victims. All were male, mostly with gunshot wounds to the head. DNA analysis and cross-matching of the samples with relatives resulted in 14 positive identifications using the Y-chromosomal STRs and 4 positive identifications using the autosomal STRs.

Conclusions

This study showed that even in cases of more than 50-year-old, highly degraded human remains from mass graves, Y-chromosomal and autosomal STRs analysis can contribute to identification of the victims.After the partisan (communist-led) forces had entered Dubrovnik on October 18, 1944, mass executions followed, among them the execution on the nearby island of Daksa (Figure 1 and ​and2).2). According to historical sources (1), the execution took place on October 25, 1944 and the following days. According to the verdict issued by the “Court of the Military Command for the South Dalmatian Region” and announced on a poster on October 29, 1944, 36 Dubrovnik citizens, most of them prominent intellectuals, were “sentenced to be shot by a firing squad.” The same historical sources do not state whether a trial was held or if there was a court under such a name (1).Open in a separate windowFigure 1Map of Croatia showing its position in Europe and Dubrovnik with its nearby island of Daksa.Open in a separate windowFigure 2Map of the island of Daksa showing two sites of the mass grave, Location 1 and Location 2.In forensics, if the recovery of skeletons takes places a long time after death the possibilities to obtain information about victims’ clothing, personal belongings, sex, age, height, and other characteristics may often be reduced. In such cases, DNA typing techniques may provide useful information. Y-short tandem repeat (STR) markers are paternally inherited and allow identification of male missing persons when the only available reference is a male paternal relative. The autosomal STR DNA profiling was established by using the commercially available miniSTR assay, which proved useful to obtain information from ancient and degraded DNA samples and was used in cases when the only available reference was a missing person’s daughter (2). The DNA technology, including both STR analysis and mitochondrial DNA analysis, was already confirmed as a method of choice in the identification of missing persons in the 1991-1995 war in Croatia (3,4).The aim of this study was describe the organization, field work, forensic anthropological examination, and DNA analysis by chromosome and autosomal STRs conducted to identify the victims from a World War II mass grave on the island of Daksa.     

Translating genomic information into clinical medicine: Lung cancer as a paradigm

We are currently in an era of rapidly expanding knowledge about the genetic landscape and architectural blueprints of various cancers. These discoveries have led to a new taxonomy of malignant diseases based upon clinically relevant molecular alterations in addition to histology or tissue of origin. The new molecularly based classification holds the promise of rational rather than empiric approaches for the treatment of cancer patients. However, the accelerated pace of discovery and the expanding number of targeted anti-cancer therapies present a significant challenge for healthcare practitioners to remain informed and up-to-date on how to apply cutting-edge discoveries into daily clinical practice. In this Perspective, we use lung cancer as a paradigm to discuss challenges related to translating genomic information into the clinic, and we present one approach we took at Vanderbilt-Ingram Cancer Center to address these challenges.Knowledge about genetic alterations that drive and sustain the growth of various cancers is exploding. Using lung cancer as a model, we describe evolving trends and challenges in leveraging genetic information to inform cancer care. We briefly review the history of chemotherapy and targeted therapies in lung cancer, with a focus on the current era of therapies targeting the protein products of driver mutations occurring in single oncogenes. We discuss challenges related to how tumors will be profiled, including issues related to who should perform testing and with what platform, who should pay for testing and how gene patents may affect costs, which tumors to profile and when, and the concept of companion diagnostics. We also discuss issues related to clinical interpretations of genomic information: how will they be reported to clinicians, in what format, and using what knowledge resources (Fig. 1). Finally, we present one approach that we took at the Vanderbilt-Ingram Cancer Center to address these issues for our personalized cancer medicine initiative.Open in a separate windowFigure 1.Complexities of genetically informed cancer medicine. As shown here, multiple factors need to be addressed in order to effectively translate knowledge of tumor gene mutations into routine clinical practice.     

Therapeutic Options for the Management of Postprandial Glucose in Patients With Type 2 Diabetes on Basal Insulin

In Brief For patients with type 2 diabetes who require add-on therapy to metformin plus basal insulin, GLP-1 receptor agonists may be a favorable option because they effectively manage postprandial glucose, reduce body weight, and have an overall favorable safety profile compared to other agents. Given the wide range of treatment combinations available for type 2 diabetes management, health professionals must partner with patients to determine the best choices based on patients’ individual lifestyle, resources, and treatment goals.Providing patients with optimal strategies for the management of hyperglycemia associated with type 2 diabetes is challenging. This is especially true as type 2 diabetes progresses and patients require two- and three-drug combinations or complex insulin regimens to achieve glycemic targets (1). Current consensus guidelines from the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD), as well as the 2015 diabetes management algorithm of the American Association of Clinical Endocrinologists, recognize that many different drug combinations can be used to achieve A1C goals (Figure 1) (1,2). Given this range of available therapeutic options, ADA/EASD guidelines emphasize the importance of individualized, patient-centered care (1). If patients are able to be involved with treatment decisions, health care professionals (HCPs) must use a shared decision-making process to increase patient satisfaction and adherence to treatment (3). HCPs should emphasize treatment outcomes that are also important to the patient (3). Factors to consider in such individualized type 2 diabetes treatment plans include patients’ attitudes and willingness to make lifestyle changes and risk factors for hypoglycemia and other adverse events. HCPs should also consider patients’ body weight, duration of disease, life expectancy, comorbidities, established vascular complications, overall level of support, and economic burdens of treatment (1). All treatment plans should include strategies for controlling obesity, blood pressure, and hyperlipidemia and emphasize smoking cessation, regular exercise, and healthy eating habits (4).Open in a separate windowFIGURE 1.ADA/EASD general recommendations for type 2 diabetes management (1). DPP-4-i, DPP-4 inhibitor; Fx’s, fractures; GLP-1-RA, GLP-1 receptor agonist; HF, heart failure; SU, sulfonylurea.^aConsider beginning at this stage in patients with a very high A1C level (e.g., ≥9%).^bConsider rapid-acting, nonsulfonylurea secretagogues (meglitinides) in patients with irregular meal schedules or who develop late postprandial hypoglycemia on sulfonylureas.^cUsually a basal insulin (NPH, glargine, or detemir) in combination with noninsulin agents.^dCertain noninsulin agents may be continued with insulin. Consider beginning at this stage if patient presents with severe hyperglycemia (≥300−350 mg/dL; A1C level ≥10.0−12.0%) with or without catabolic features (e.g., weight loss or ketosis).     

Cupid: simultaneous reconstruction of microRNA-target and ceRNA networks

We introduce a method for simultaneous prediction of microRNA–target interactions and their mediated competitive endogenous RNA (ceRNA) interactions. Using high-throughput validation assays in breast cancer cell lines, we show that our integrative approach significantly improves on microRNA–target prediction accuracy as assessed by both mRNA and protein level measurements. Our biochemical assays support nearly 500 microRNA–target interactions with evidence for regulation in breast cancer tumors. Moreover, these assays constitute the most extensive validation platform for computationally inferred networks of microRNA–target interactions in breast cancer tumors, providing a useful benchmark to ascertain future improvements.MicroRNAs (miRNAs) regulate RNA stability and mRNA translation (Filipowicz et al. 2008) and their dysregulation has been implicated in a wide range of human diseases including cancer (Garzon et al. 2009). Consequently, establishing accurate and comprehensive repertoires of miRNA–target interactions is a necessary step toward elucidating their mechanistic role in pathophysiology. Dissecting miRNA regulation, however, has proven challenging because candidate miRNA binding sites are ubiquitous and their regulatory effects are context specific (Liu et al. 2005; Lu et al. 2005; Mukherji et al. 2011). As a result, and despite their relatively low accuracy, computational prediction methods that incorporate context-specific data are preferred for screening for miRNA–target interactions in tumor contexts (Carroll et al. 2013; Erhard et al. 2014).To address these challenges, we introduce Cupid, an integrative framework for the context-specific inference of miRNA targets. Cupid integrates sequence-based evidence and functional clues derived from RNA and miRNA expression analysis, predicting candidate miRNA binding sites and associated target genes using ensemble machine learning classifiers that are trained on validated interactions. Candidate interactions emerging from this step are then refined based on independent, context-specific clues, including their predicted ability to mediate competitive endogenous RNA (ceRNA) interactions, where mRNA compete for shared miRNA regulators (Fig. 1A; Tay et al. 2014). Thus, Cupid simultaneously infers both interaction types (ceRNA and miRNA–target interactions). In addition, we considered evidence for combinatorial regulation by multiple miRNA species (Fig. 1B; Boissonneault et al. 2009; Xu et al. 2011) and for indirect miRNA regulation through effector proteins (Fig. 1C). Taken individually, these clues are predictive of bona fide miRNA–target interactions and can significantly improve the tradeoff between precision and recall.Open in a separate windowFigure 1.Methodology. (A) Cupid first reevaluates sites predicted by TargetScan, miRanda, and PITA, selecting and rescoring each candidate site (Step I). Sites are used to select and score miRNA-target interactions (Step II), which are then examined for evidence for mediating ceRNA interactions (Step III). In addition, to support interaction prediction we considered (B) evidence for combinatorial regulation between miRNAs and (C) evidence for indirect regulation by miRNAs through effectors. (D) The majority of site predictions by TargetScan, miRanda, and PITA are exclusive to a single algorithm; for example, <10% of sites predicted by miRanda are also predicted by another method. (E) Cupid predicted 529K miRNA-target interactions in Step II, excluding 60% of Step I candidate interactions. As a result, it makes considerably fewer predictions than TargetScan, miRanda, and PITA. (F) Cupid Step III predictions include less than a quarter of Step I candidate interactions.We show that Cupid predictions outperform other leading algorithms, based on multiple experimental assays, including PAR-CLIP data, miRNA perturbation followed by mRNA and protein expression profiles, and 3′ luciferase activity assays. Critically, while Cupid predicts fewer interactions than other methods (Fig. 1D–F), its predictions are much more likely to be consistent with experimental evidence. This is critical since high false-positive prediction rates are a key limitation of current miRNA–target prediction methods.     

Immunization of Aotus Monkeys with Recombinant Plasmodium falciparum Hybrid Proteins Does Not Reproducibly Result in Protection from Malaria Infection

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.The increasing drug resistance of Plasmodium falciparum, the most pathogenic human malaria parasite, underlines the need for an effective malaria vaccine. Identification, testing, and optimization of candidate molecules originating from all developmental stages of the parasite are under way. Previously, a successful trial in Aotus monkeys employed the Escherichia coli-expressed hybrid proteins MS2/SERP/HRPII and SERP/MSAI/HRPII (11). Both hybrid proteins contain a region of the serine repeat protein SERP (1, 9), including two putative T-cell epitopes (13) and previously shown to induce a partial protective response in Aotus monkeys (5), and the C-terminal half of the histidine-rich protein HRPII (14), which has also been shown to induce a partially protective response (5, 8). SERP/MSAI/HRPII contains in addition a conserved N-terminal region of the merozoite surface antigen MSAI (7) that includes at least four T-cell epitopes (3, 6). Here we report on further analysis of three hybrid proteins of this type in a vaccination trial with Aotus monkeys. Two of the proteins, SERP/HRPII and SERP/MSAI/HRPII, are improved versions of the hybrid proteins mentioned above, obtained by deleting nonmalaria protein regions and changing an internal restart residue (methionine-729 of SERP) into alanine. Thus, the SERP/HRPII hybrid protein comprises residues 630 to 893 of SERP fused to the 189 C-terminal residues of HRPII, and SERP/MSAI/ HRPII comprises residues 630 to 764 of SERP fused to residues 146 to 259 of MSAI, which is fused to the 189 C-terminal residues of HRPII. SERP/41-3/HRPII contains the same components as SERP/HRPII, and additionally includes residues 77 to 188 of antigen 41-3 (10), which was previously shown to confer protection against a P. falciparum challenge (5). The internal restart residue (methionine-100) was also mutagenized into alanine and another residue, arginine-319, was changed into leucine to prevent proteolytic degradation. SERP/MSAI/HRPII was partially purified to a final purity of about 30%, as described previously (8), in order to match the quality of the proteins used in the successful previous trials (5, 11). The other two hybrid proteins were purified from bacterial lysates to over 90% purity by size exclusion chromatography (SERP/41-3/HRPII) or by sequential cation and anion exchange and then size exclusion chromatography (SERP/ HRPII) (data not shown). The final products were dialyzed against phosphate-buffered saline–3 M urea and adjusted to 100 μg of protein per ml. Efficacy was tested following an experimental protocol identical to the one used in the previous successful trial (11).Fifteen laboratory-raised Aotus azarae boliviensis karyotype VI monkeys were randomly assigned to one of four experimental groups (three groups of four and one group of three monkeys) and immunized with 1 ml of antigen or with the diluent alone (control group), both mixed with 100 μl of polyalphaolefin (4) as an adjuvant, on days 0, 21, and 42. Each vaccine dose was administered subcutaneously at two separate sites in the right and left flank and was well tolerated. The seroconversion results, as measured by enzyme-linked immunosorbent assay with SERP/HRPII as the solid-phase antigen and peroxidase-labelled rabbit anti-human immunoglobulin G (1:10,000 dilution; Pierce) as the secondary antibody, are shown in Fig. ​Fig.1.1. All experimental monkeys developed comparable antibody responses to SERP/HRPII, irrespective of the immunogen. Control monkey sera did not react significantly (not shown). A boosting effect is obvious after the second injection in all three groups (Fig. ​(Fig.1),1), as well as after the third SERP/41-3/HRPII injection (Fig. ​(Fig.1C).1C). This is similar to the seroconversion pattern observed previously (11). Prechallenge sera were also tested by immunofluorescence (IFA) for reactivity with P. falciparum schizonts. All preimmune sera and control group immune sera were negative (1:100 dilution). IFA titers from the experimental animals were all 1:1,600, except for animals A381 and A462 (titer, 1:800) and A452 and A292 (titer, 1:3,200). Thus, antibodies specific for native parasite determinants were induced. The relatively low IFA titers were comparable to those obtained in previous successful trials (5, 11). Open in a separate windowFIG. 1Development of antibody responses in Aotus monkeys during the immunization period as determined by enzyme-linked immunosorbent assay. Monkeys in different immunization groups were immunized at weeks 0, 3, and 6 (indicated by arrows) and challenged at week 8 (indicated by an asterisk). All sera were tested for reactivity with SERP/HRPII in a 1:100 dilution. The hybrid antigens used for immunization were SERP/MSAI/HRPII (A), SERP/HRPII (B), and SERP/41-3/HRPII (C). Sera of the three control monkeys remained negative in this assay (not shown). OD, optical density.At week 7 all monkeys were splenectomized, and at week 8 they received intravenously 2 × 10⁶ parasitized erythrocytes, which had been isolated from an Aotus monkey infected with an in vivo-passaged FUP-Cayenne isolate of P. falciparum (a kind gift of W. E. Collins) (Fig. ​(Fig.1).1). Monkey A293 appeared to have no spleen, although there was no prior history of splenectomy. The immunoglobulin G response of monkey A293 was nevertheless comparable to that of the other animals (Fig. ​(Fig.1B).1B). Figure ​Figure22 shows the course of parasitemia after challenge. Two of three control animals rapidly developed a parasitemia which required mefloquine therapy (20 mg/kg of body weight orally) when parasitemia reached 10% (day 8 for A371 and day 10 for A320). In A432, parasitemia developed to 8.6% (day 10) and then fluctuated until rapidly reaching 19% (day 21), at which point mefloquine was administered (Fig. ​(Fig.2A).2A). None of the three immunized groups showed a solid protective response (Fig. ​(Fig.2B2B to D). A340 (SERP/HRPII group) (Fig. ​(Fig.2C)2C) and A292 (SERP/41-3/HRPII group) (Fig. ​(Fig.2D)2D) showed low fluctuating parasitemias with a peak around 2.5% at the end of the observation period (day 25). Otherwise, parasitemias of the experimental animals did not significantly differ from those of the controls. No obvious correlation between prechallenge antibody levels and protection was evident. Open in a separate windowFIG. 2Course of infection with the FUP-Cayenne isolate of P. falciparum in control Aotus monkeys (A) and in Aotus monkeys immunized with SERP/MSAI/HRPII (B), SERP/HRPII (C), and SERP/41-3/HRPII (D). Parasitemias of ≥10% were cured with mefloquine.It seems unlikely that small conservative changes designed to improve SERP/HRPII and SERP/MSAI/HRPII expression in E. coli and to remove nonrelevant sequences adversely affected immune response development. After challenge, parasitemia developed markedly faster than in the previous trial, which had shown protection. Challenge with 2 × 10⁶ parasitized erythrocytes now resulted in high parasitemias on days 7 to 9 in 2 of the 3 controls and in 6 of the 12 experimental animals, whereas previously controls were untreated until day 14 (11). Also, one control and three experimental animals suffered recrudescence, which was not seen previously (11) or with later infections with the same parasite stock (5). It is remarkable that this apparent enhanced pathogenicity developed after a single passage in A. nancymai just before the present trial started. It is likely that this unintended pathogenicity influenced the experimental outcome. The protection of two monkeys in the SERP/HRPII and SERP/41-3/HRPII group may, however, reveal some protective effect of these vaccine candidates.For demonstration of the protective potential of antigens in the primate model the pathogenicity of the challenge strain in the respective primate (sub)species, i.e., the equilibrium of immune response and pathogenicity, seems to be crucial (2, 12). The disturbance of this equilibrium may explain the discrepancy between previous successful trials (5, 11) and the present study. Recombinant proteins shown to be protective in the Aotus model (5, 11) failed to protect Saimiri monkeys, in which the course of parasitemia is quite different from that observed in Aotus monkeys. Similarly, no protection could be demonstrated in A. nancymai against the same challenge strain as was used in the successful trials with A. azarae boliviensis and A. lemurinus griseimembra (7a). The poor standardization of these models due to the scarcity of monkeys susceptible to human malaria remains an obstacle for the evaluation of human malaria vaccine candidates.     

Relationship between the Clinical Characteristics and Intervention Scores of Infants with Apparent Life-threatening Events

We investigated the clinical presentations, diagnostic and therapeutic modalities, and prognosis from follow-up of infants with apparent life-threatening events (ALTE). In addition, the relationship between the clinical characteristics of patients and significant intervention scores was analyzed. We enrolled patients younger than 12 months who were diagnosed with ALTE from January 2005 to December 2012. There were 29 ALTE infants with a peak incidence of age younger than 1 month (48.3%). The most common symptoms for ALTE diagnosis were apnea (69.0%) and color change (58.6%). Eleven patients appeared normal upon arrival at hospital but 2 patients required cardiopulmonary resuscitation during the initial ALTE. The most common ALTE cause was respiratory disease, including respiratory infection and upper airway anomalies (44.8%). There were 20 cases of repeat ALTE and 2 cases of death during hospitalization. Four patients (15.4%) experienced recurrence of ALTE after discharge and 4 patients (15.4%) showed developmental abnormalities during the follow-up period. The patients with ALTE during sleep had lower significant intervention scores (P=0.015) compared to patients with ALTE during wakefulness and patients with previous respiratory symptoms had higher significant intervention scores (P=0.013) than those without previous respiratory symptoms. Although not statistically significant, there was a weak positive correlation between the patient''s total ALTE criteria and total significant intervention score (Fig. 2, r=0.330, P=0.080). We recommend that all ALTE infants undergo inpatient observation and evaluations with at least 24 hr of cardiorespiratory monitoring, and should follow up at least within a month after discharge.Open in a separate windowFig. 2Significant intervention scores according to ALTE situation and recent URI history. The patients with ALTE during sleep had significantly lower scores (P = 0.015) compared to patients with ALTE during wakefulness, and the patients with previous respiratory symptoms had significantly higher scores (P = 0.013) than those without previous respiratory symptoms.

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