Tuesday, 23 January 2018

Nanotechnology- A Promising Approach for Suicide Gene Therapy


Cancer is one of the world’s most dreadful diseases and the battle against cancer continues till date. Suicide gene therapy for cancer is one of the best approaches for annihilation of cancer. In brief, suicide gene codes for an enzyme which converts a nontoxic prodrug into toxic metabolites and subsequently mediates death of host cells itself on account of which it is named “suicide” gene therapy. These suicide gene when constitutively expressed by the cells not only mediates death of host cells but also inflicts strong bystander effects on neighboring cells by predisposing them to toxic downstream metabolites. Due to such advantages, they manifest minimal systemic toxicity and are also effective against many drug resistance cancer cells. Among all existing suicide genes, Cytosine Deaminase (CD) and Herpes Simplex Virus-thymidine kinase (HSVtk) have shown promising results initially and has been investigated extensively since long. The HSVtk enzyme initially phosphorylates the prodrug Ganciclovir (GCV) to its monophosphate form, which is subsequently phosphorylated again by endogenous cellular kinase to generate nucleotide analogs (di- and triphosphate forms of GVC). Triphosphate form of GCV is then readily incorporated into DNA during the course of DNA synthesis and acts as a chain terminator to prevent further DNA synthesis, which ultimately induces cell death.
The therapeutic efficacy of HSVtk suicide gene therapy is often limited by cell-to-cell contact which is a prerequisite for transport of downstream metabolic byproducts of ganciclovir to neighboring cells so as to attain bystander-killing effect. As an outcome of such drawbacks, HSVtk suicide gene does not seem to be effective against different cell types. In contrary to this, Cytosine Deaminase (CD) efficiently converts prodrug 5-Fluorocytosine (5- FC) into therapeutically active anticancer agent 5-Fluorouracil (5- FU), which subsequently permeates across the cell membrane to mediate bystander killing effects on adjacent neighboring cells. Thus, 5-FC/CD system attains suicide gene therapy much more efficiently as compared to other counterparts. Although 5-FC/CD system attains better therapeutic outcomes, it is ineffective against 5-FC resistant cancer cells and thus its anticancer potential could not be generalized for all cancer types. In order to overcome such drawback, Gopinath et al. have designed Cytosine Deaminase-Uracil Phosphoribosyltransferase (CD-UPRT) bifunctional suicide gene construct in which Uracil Phosphoribosyltransferase (UPRT) acts upon product of CD i.e. 5-FU and converts it further into other toxic metabolites.
The therapeutic effect of suicide genes can be enhanced by combinatorial approaches. In combination therapy, two or more drugs with similar or different mode of action are employed to realize synergistic anticancer therapeutic potentials. Such synergistic anticancer potential of combination of radiation therapy and 5-FC/ CD plus UPRT gene therapy was demonstrated by Kambara et al. against malignant gliomas [8]. Apart from this, the combination therapy also provides scope for exploiting radio sensitizing properties of 5-FU and by stander effects during the course of treatment. Many research groups have reported the use of suicide gene in combination with chemotherapy and radiation to enhance the therapeutic effect and to overcome the drug resistance. Gopinath et al. were the first to report the applications of silver nanoparticles for synergizing the therapeutic effect of suicide gene. They have also reported the synergistic therapeutic effect of suicide gene with anticancer drug curcumin. One of the major challenging tasks in suicide gene therapy is lack of suitable vectors for targeted delivery of suicide gene to cancer cells. The application of such DNAbased therapeutics is largely limited due to poor cellular uptake, degradation by serum nucleases and rapid renal clearance following systemic administration. In addition to these, organ specific targeted DNA therapy has been a major challenge to overcome off-target gene therapy. In order to circumvent these limitations, numerous organ specific targeted nanocarriers have been developed recently for systemic administration.

Monday, 22 January 2018

Bedside Ocular Ultrasound for Diagnosis of Visual Disturbances in Hypertensive Disorders of Pregnancy


A 41year-old woman in her first pregnancy attended our emergency department due to high blood pressure, headaches and visual disturbances. She was taken to the operating room for an emergency caesarean section. In postoperative she developed HELLP syndrome. Due to the persistence of the headaches and visual disturbances, an ocular ultrasound was performed which showed, in both eyes, a laminar hyperechogenic image in the vitreous cavity compatible with bilateral serous retinal detachment (Figure 1). Measurement of the diameter of the optic nerve sheath was similar in both eyes, 5, 4mm in the right eye and 5, 5mm in the left eye, which was indicative of increased intracranial pressure [1-4]. A Cranial CT scan was performed and was normal. In the following days, blood pressure was controlled and the headaches and visual disturbances were no longer present. An ocular ultrasound no longer showed images of retinal detachment nor increased intracranial pressure (Figure 2). We believe that bedside ocular ultrasound is a useful tool in hypertensive disorders that are associated with visual disturbances.

Friday, 19 January 2018

Comparison of Local versus General Anaesthesia for Carpal Tunnel Release


Carpal tunnel syndrome (CTS) is a common neuropathy, affecting the median nerve as it passes under the transverse carpal ligament. CTS is described as a nerve compression at the wrist plane, CTS causes numbness and tingling in the hand and fingers. Sir James Paget defined CTS first in1853, and since then, in the 1950s a scientist named George Phalen popularized the diagnosis and treatment of CTS. The aetiology of CTS is considered idiopathic in most cases but it is still controversial. Conservative treatment consists of splinting or corticosteroid injections and surgical release of the carpal tunnel are the treatment method options. There has been continued debate over the optimal management of this disease. Decision of the surgeon has consistently varied.
Carpal tunnel release (CTR) is known as an effective treatment for idiopathic CTS. CTR is performed with a variety of techniques such as endoscopic (ECTR) or open (OCTR). Literature has not got consensus on the superiority of any one technique to another. Local anaesthesia (LA) and general anaesthesia (GA) are anaesthetic options on the surgical treatment of CTS. LA is safe, fast, and effective, but the injection could be painful. In one recent series, about 10% of patients indicated that they would prefer another form of anaesthesia. When applying the local anaesthetics under the skin patient could have pain and pain could make the patient uncomfortable. Also from the surgeon side discomfort of the patient could affect the surgical procedure and sometimes visualization of the surgical area could be difficult due to oedema caused by local anaesthetics. These problems in surgical procedure could affect the clinical results of the surgery. Sedation or GA could make the procedure more comfortable.
The aim of the present study was to compare the effects of the anaesthetic methods on the patient’s clinical results. To our knowledge this is the first report that compares the clinical outcomes of the open CTR with GA or LA. Computerized patient database was searched to identify all patients with CTS who underwent open CTR between January 2009 and January 2013 at Fatih Sultan Mehmet Training and Research Hospital. The year 2013 was selected to ensure a minimum one-year follow-up. At the result of the search total of 148 patients (169 CTS hands) were found. Of the 148 patients (169 CTS hands) 21 patients were operated bilaterally and excluded from study. Of the 127 patient operated monolaterally. One patient died from another reason and 14 patients was not available and excluded from the study. Fifty five patients operated with GA (group A) and 57 patients with LA (group B). The last available 50 monolateral patient operated by the same surgeon for each group included to the study.

Thursday, 18 January 2018

Fronto-Temporal Dementia: A Case Report


Fronto-temporal dementia (FTD) is the most common form of primary degenerative dementia after Alzheimer’s disease that affects middle age with an estimated prevalence at 15 per 100,000 in the population aged between 45 – 65 years. FTD is often misdiagnosed due to its early onset, clinical and pathological heterogeneity. Neuroimaging is known to assist in early diagnosis of these disorders. We present a case of a 59 year old woman with FTD.
A 59 year old female was diagnosed with major depressive disorder and borderline personality disorder of about a year’s duration. There were continued behavioral changes, with periods of aggression and impulsiveness at times. Also, there was ongoing worsening of memory with poor concentration. She later developed irritable mood and poor appetite. Examination on her last hospital visit showed a well groomed, alert, calm and cooperative patient. She had normal speech and normal psychomotor activity. Her mood was labile; she was however coherent and relevant showing reactive affects. She was a suicidal however objectively hallucinating.
The patient’s medications included fluoxetine 20mg daily, sodium valproate 200mg twice daily, clonazepam 0.5mg when necessary. She underwent magnetic resonance imaging (MRI) of the brain in view of her worsening memory and concentration. This showed evidence of mild frontotemporal lobe involution (Figure 1). Incidentally, there was also an enlarged anterior pituitary gland (1.02 x 1.3 cm) seen.
She was also referred to the nuclear medicine department for brain perfusion imaging – this forms part of molecular imaging and was done byacquiring SPECT images of the brain using Tc-99m HMPAO (Exametazime) injected into a pre-inserted IV line in a quiet dimly lit room. The scan showed a relatively decreased perfusion in the frontotemporal region, involving the anterior cingulate and anterior temporal cortices seen in Figure 2. These findings were in agreement with the findings of MRI and together with the clinical history and signs, consistent with frontotemporal dementia with a diagnosis of Pick’s disease most likely.
The concept of frontotemporal dementia includes a group of primary degenerative dementia disorders that presents with predominant frontal lobe and /or temporal lobe symptoms. These include Pick’s disease (often referred to in broader term of frontotemporal dementia), frontotemporal lobar degeneration (FTLD) also called dementia lacking distinctive histological features (DLDH), frontotemporal lobar degeneration with motor neuron disease, corticobasal degeneration, progressive supranuclear palsy, frontotemporal dementia with parkinsonism linked to chromosome 17 and others.

Wednesday, 17 January 2018

Darwinian Factors in the Clinical Expression of Multiple Sclerosis

Many predisposing factors for multiplesclerosis (MS), such as HLA types and geomagnetic fields have been described but the search for a single essential factor has been like searching for the rainbow’s end. The most notable feature in the epidemiology of MS in the Western world has been its rise from unknown to the most prevalent disabling neurodegenerative disease of young adults. We suggest that this may be largely or entirely attributable to societal changes that have increasingly isolated populations from micro-organisms that form part of the human microbiome and which are essential for an effective maturation of immune defence mechanisms. This Darwinian explanation suggests a rational approach to both prevention and treatment of MS, by substituting for the loss or absence of factors that millions of years of evolution have led the immune system to ‘expect’ to encounter early in life.
BCG: Bacille Calmette-Guérin; CDMS: Clinically Definite Multiple Sclerosis; CIS: Clinically Isolated Syndrome; EBV: Epstein- Barr virus; HERV: Human Endogenous Retrovirus; HLA: Human Leucocyte Antigen; IL: Interleukin; INF-α: Interferon Alpha; MAIT: Mucosal-associated Invariant T Cells; MAMP: Microbe-associated Molecular Pattern; MHC: Major Histocompatibility Complex; MRI: Magnetic Resonance Imaging; MS: Multiple Sclerosis; TCR: T Cell Antigen Receptor; TNF-α: Tumour Necrosis Factor Alpha; Treg: Regulatory T Cell; PRR: Pattern Recognition Receptor; TLR: Toll-like Receptor; RA: Retinoic Acid.
Current treatment strategies for multiple sclerosis (MS) are essentially empirical because no single clear underlying cause of the disease has been determined. Numerous genomic and environmental risk factors for MS have been described, but problems of cause and effect remain unresolved. It has indeed been stated, with considerable justification, that MS research is “low on fact, high on fiction”.
Although there is a well-described genetically determined predisposition to MS, the association is far from complete as, for example, the risk of an identical twin sibling of an affected person developing MS is only 30%. Thus any genes involved are likely to be of low penetration and influenced by the expression of some or many other genes. Notwithstanding, a large number of genetic loci, including many coding for HLA, have been determined and fine-mapped. These studies have demonstrated a central role for the immune system in the aetiopathogenesis of MS but the highly complex nature of the data will require novel tools for their analysis before any unifying factor can be delineated.

Tuesday, 16 January 2018

Intersection of Apoptosis and Autophagy Cell Death Pathways


The balance between cell survival and death is a critical parameter in the regulation of cell and tissue homeostasis. Autophagy is an evolutionarily conserved mechanism for the gross disposal and recycling of intracellular proteins in mammalian cells. Autophagy also kills cells under certain conditions, in a process called autophagic cell death; this involves pathways and mediators different from those of apoptosis. Therefore, three different mechanisms of cell death have been identified in mammalian cells; namely, apoptosis (type I), autophagic cell death (type II), and necrosis (type III). Whether and how these different processes of cell death interconnected each other has not been fully clarified. In this review we discuss the evidence supporting a mechanistic link especially focusing between apoptosis and autophagy associated cell death—including the possibility of cross–talk between the relevant signaling pathways—that could serve to maintain cellular homeostasis in mammals.
In recent decades, insight into the molecular regulation of autophagy in mammalian cells has come from the discovery and functional analysis of Autophagy-Related Gene (ATG). Autophagy is an evolutionally conserved homeostatic process for intracellular degradation by which intracellular proteins are sequestered in a double–membrane–bound autophagosome and delivered to the lysosome during stress conditions; this process facilitates both degradation and recycling of intracellular proteins in mammalian cells. The molecular machinery of autophagy co-ordinates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. Defective autophagy underlies a wide variety of human disease and physiology including cancer, neurodegeneration, and infectious diseases. Mammalian orthologues of ATG family proteins have been identified and various functions of ATG proteins have been elucidated, including how these proteins control the formation of autophagosomes. Although autophagy was originally characterized as a cytoprotective process in yeast under starvation conditions, it is now thought to be a form of cell death along with the two classical mechanisms of apoptosis and necrosis in mammalian cells.
Three possible mechanisms for cell death have been known to exist in mammalian cells, namely apoptosis (type I cell death), autophagic cell death (type II cell death), and necrosis (type III cell death). Apoptotic cell death (type I cell death) is characterized by rounding up of the cell and reduction of cell volume, chromatin condensation, nuclear fragmentation, no modification of cytoplasmic organelle, and plasma membrane blebbing without involvement of gene activity. Since autophagy is thought to be a pro-survival pathway, whether or not autophagy indeed 

Friday, 12 January 2018

Metallo-Beta-Lactamase Producing Gram-Negative Bacteria among Patients Visiting Shahid Gangalal National Heart Centre


The rapid spread of acquired Metallo-Beta-Lactamases (MBL) among major Gram-negative pathogens is an emerging threat and a matter of concern worldwide as it results into fewer therapeutic options for the treatment. Therefore, this study was undertaken to determine the prevalence of MBL producing Gram-negative bacteria isolated from different clinical samples.
A total of 490 samples were analyzed, at the Microbiology Department of Shahid Gangalal National Heart Centre (SGNHC), Bansbari, Kathmandu from December 2013 to June 2014, for routine culture and antibiotic susceptibility testing. MBL detection was done by Imipenem-EDTA Combined Disc Test.
Out of 490 samples analyzed, 107 showed positive growth. Fortytwo percent of the Gram-negative isolates were Multi Drug Resistant (MDR). Among 107 Gram-negative isolates, 66 ceftazidime resistant isolates were screened for MBL production of which 9 (13.6%) were found to be MBL positive. All MBL positive isolates were Pseudomonasaeruginosa. None other Gramnegative bacteria were found to produce MBL. Prevalence of MBL producing P. aeruginosa was 20% and all the isolates were MDR. All the MBL producing P. aeruginosa were isolated from hospitalized patients.
This study showed MBL production in a considerable number of P. aeruginosa isolates with MDR phenotypes. There is a need to track the detection of MBL producers and judicious use of carbapenems is necessary to prevent the further spread of these organisms.
AST: Antibiotic Susceptibility Test; ATCC: American Type Culture Collection; CLSI: Clinical Laboratory Standard Institute; EDTA: Ethylene-Diamine-Tetraacetic Acid; ESBL: Extended- Spectrum-Beta-Lactamase; ICU: Intensive Care Unit; MBL: Metallo- Beta-Lactamase (Metallo-β-lactamase); MBLs: Metallo-Beta- Lactamases; MDR: Multi-Drug Resistance; SPSS: Statistical Package for Social Science; TUTH: Tribhuvan University Teaching Hospital; ZOI: Zone of Inhibition.