X-linked hyper-immunoglobulin M syndrome harboring a novel CD40-ligand gene mutation: a case report.

The X-linked hyper-IgM syndrome (X-HIGM1) is a rare primary immunodeficiency disorder (PID) caused by mutations in the gene encoding the CD154 protein, also known as CD40 ligand (CD40LG). X-HIGM1 is characterized by normal or elevated serum levels of IgM in association with decreased levels of IgG, IgA, and IgE. The CD40LG protein expressed on activated T cells interacts with its receptor protein, CD40, on B lymphocytes and dendritic cells. Mutations in the CD40LG gene lead to the production of an abnormal CD40L protein that fails to attach to its receptor, CD40 on B cells resulting in failure to produce IgG, IgA, and IgE antibodies. In the present study, we investigated the molecular defects underlying such a PID in a patient presenting with clinical history of pneumonia and acute respiratory distress syndrome (ARDS) at 7 months of age and diagnosed as transient hypogammaglobulinemia with decreased levels of IgG and increased levels of IgM. We have identified a novel and yet to be reported frame shift deletion of a single base pair (c.229delA) in exon 2 (p.Arg77AspfsTer6) of the CD40L gene ensuing the premature truncation of the protein by 6 amino acids by targeted gene sequencing. This frame shift mutation identified as a CD40L variant was found to be pathogenic which was also validated by Sanger sequencing. The in-silico analysis of c.229 del A mutation also predicted the change to be pathological affecting the structure and function of the CD40L (CD40L, CD154) protein and its protein-protein interaction properties.

Childhood Early T Cell Precursor Acute Lymphoblastic Leukaemia with t(12;17) (p13;q21) Translocation - A Rare Entity or Part of ETP/Myeloid Mixed Phenotype Acute Leukaemia.

The translocation t (12;17) (p13; q21) is a rare cytogenetic event most commonly described in pre-B- acute lymphoblastic leukaemia and acute myeloid leukaemia. We identified a child with an immunophenotype of Early T Cell Precursor Acute Lymphoblastic Leukaemia ETP- ALL having t (12;17) (p13; q21) translocation as the primary karyotypic anomaly. The association of t (12;17) (p13; q21) with ETP-ALL has not been described previously in literature. The possibility of it being a novel genetic abnormality or a part of the newly described entity of ETP/myeloid MPAL is being discussed. Detection of such abnormalities can alter the prognosis of ETP-ALL. Key words: ETP-ALL , t(12;17) (p13;q21) translocation, ETP- MPAL.

Predicting transport of intra-articularly injected growth factor fusion proteins into human knee joint cartilage.

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application. STATEMENT OF SIGNIFICANCE: The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application.

Consumption Coagulopathy in Paediatric Solid Tumours: A Retrospective Analysis and Review of Literature.

INTRODUCTION: Disseminated intravascular coagulation or consumption coagulopathy is a well-recognized entity both in various malignant and non-malignant conditions. Most data in paediatric solid tumours are isolated case reports, while there is sparse data in paediatric acute leukaemia. OBJECTIVE: The study was conducted to analyze the incidence of DIC in our population of paediatric solid tumours. DESIGN: All records of children <15 years of age registered in the Paediatric Oncology department of our institute with a diagnosis of solid tumour malignancy were retrospectively reviewed for evidence of DIC. RESULTS: Out of the 73 children, 4 have developed DIC, an incidence of 5.5%. The mean age of children who developed DIC was 4.6 years (Range- 2months -15 years) and the majority (2/4- 50%) children were less than 1 year of age. Children with DIC had a male predominance (75%) and the majority (75%) presented in advanced stages of the disease. Of the 10 children with neuroblastoma, 2 (20%) had evidence of DIC. Statistical analysis was done to determine whether any patient characteristic had the propensity to develop DIC. The only factor that attained statistical significance was younger age. CONCLUSION: Disseminated intravascular coagulation though uncommon in children should always be thought of in a child with advanced disease presenting with thrombocytopenia or clinical manifestations of bleeding tendency. An index of suspicion is important for early diagnosis and emergent treatment which eventually improves survival.

Green fluorescent proteins engineered for cartilage-targeted drug delivery: Insights for transport into highly charged avascular tissues.

Osteoarthritis (OA), the most common form of arthritis, is a multi-factorial disease that primarily affects cartilage as well as other joint tissues such as subchondral bone. The lack of effective drug delivery, due to the avascular nature of cartilage and the rapid clearance of intra-articularly delivered drugs via the synovium, remains a major challenge in the development of disease modifying drugs for OA. Cationic delivery carriers can significantly enhance the uptake, penetration and retention of drugs in cartilage by interacting with negatively charged matrix proteoglycans. In this study, we used "supercharged" green fluorescent proteins (GFPs), engineered to have a wide range of net positive charge and surface charge distributions, to characterize the effects of carrier charge on transport into cartilage in isolation of other factors such as carrier size and shape. We quantified the uptake, extent of cartilage penetration and cellular uptake of the GFP variants into living human knee cartilage and bovine cartilage explants. Based on these results, we identified optimal net charges of GFP carriers for potential drug targets located within cartilage extracellular matrix as well as the resident live chondrocytes. These cationic GFPs did not have adverse effects on cartilage in terms of measured cell viability and metabolism, cartilage cell biosynthesis and matrix degradation at doses needed for drug delivery. In addition to quantifying the kinetics of GFP uptake, we developed a predictive mathematical model for transport of the GFP variants that exhibited the highest uptake and penetration into cartilage. This model was further used to predict the transport behavior of GFPs during scale-up to in vivo applications such as intra-articular injection into human knees. The insights gained from this study set the stage for development of cartilage-targeted delivery systems to prevent cartilage degeneration, improve tissue regeneration and reduce inflammation that may cause degradation of other joint tissues affected by OA.

Cartilage diseases.

Hyaline cartilages, fibrocartilages and elastic cartilages play multiple roles in the human body including bearing loads in articular joints and intervertebral discs, providing joint lubrication, forming the external ears and nose, supporting the trachea, and forming the long bones during development and growth. The structure and organization of cartilage's extracellular matrix (ECM) are the primary determinants of normal function. Most diseases involving cartilage lead to dramatic changes in the ECM which can govern disease progression (e.g., in osteoarthritis), cause the main symptoms of the disease (e.g., dwarfism caused by genetically inherited mutations) or occur as collateral damage in pathological processes occurring in other nearby tissues (e.g., osteochondritis dissecans and inflammatory arthropathies). Challenges associated with cartilage diseases include poor understanding of the etiology and pathogenesis, delayed diagnoses due to the aneural nature of the tissue and drug delivery challenges due to the avascular nature of adult cartilages. This narrative review provides an overview of the clinical and pathological features as well as current treatment options available for various cartilage diseases. Late breaking advances are also described in the quest for development and delivery of effective disease modifying drugs for cartilage diseases including osteoarthritis, the most common form of arthritis that affects hundreds of millions of people worldwide.

Synthetic nanoscale electrostatic particles as growth factor carriers for cartilage repair.

The efficient transport of biological therapeutic materials to target tissues within the body is critical to their efficacy. In cartilage tissue, the lack of blood vessels prevents the entry of systemically administered drugs at therapeutic levels. Within the articulating joint complex, the dense and highly charged extracellular matrix (ECM) hinders the transport of locally administered therapeutic molecules. Consequently, cartilage injury is difficult to treat and frequently results in debilitating osteoarthritis. Here we show a generalizable approach in which the electrostatic assembly of synthetic polypeptides and a protein, insulin-like growth factor-1 (IGF-1), can be used as an early interventional therapy to treat injury to the cartilage. We demonstrated that poly(glutamic acid) and poly(arginine) associated with the IGF-1 via electrostatic interactions, forming a net charged nanoscale polyelectrolyte complex (nanoplex). We observed that the nanoplex diffused into cartilage plugs in vitro and stimulated ECM production. In vivo, we monitored the transport, retention and therapeutic efficacy of the nanoplex in an established rat model of cartilage injury. A single therapeutic dose, when administered within 48 hours of the injury, conferred protection against cartilage degradation and controlled interleukin-1 (IL-1) mediated inflammation. IGF-1 contained in the nanoplex was detected in the joint space for up to 4 weeks following administration and retained bioactivity. The results indicate the potential of this approach as an early intervention therapy following joint injury to delay or even entirely prevent the onset of osteoarthritis.

Analysis of neoadjuvant therapies in breast cancer with respect to pathological complete response, disease-free survival and overall survival: 15 years follow-up data from Kuwait.

AIMS: Optimizing neoadjuvant chemotherapy regimens is essential for achieving maximal pathological complete response (pCR) in patients with breast cancer. pCR is usually considered as a surrogate marker for survival. The aim of this study was to analyze pCR with respect to various neoadjuvant regimens and its effect on survival. METHODS: This retrospective analysis included 377 patients with stages II and III breast cancer treated between 1998 and 2009 with neoadjuvant chemotherapy. Neoadjuvant regimens were analyzed with respect to pCR, disease-free survival (DFS) and overall survival (OS). RESULTS: The median age of our population was 50 years with the majority being premenopausal and locally advanced. The overall pCR rate was 13.7% with higher rates seen in patients receiving combination of anthracyclines and taxanes (14.2%). The practice of sandwiching surgery and chemotherapy was inferior to true neoadjuvant chemotherapy of eight cycles. Addition of trastuzumab to Her2 positive patients resulted in higher pCR rates (P = 0.006). Achievement of pCR with neoadjuvant chemotherapy resulted in significantly higher DFS and OS. CONCLUSION: pCR is associated with better survival in breast cancer patients receiving neoadjuvant chemotherapy. Initial anthracycline-based chemotherapy followed by non-cross-resistant taxane-based chemotherapy along with the addition of trastuzumab in Her2 positive patients might be the optimal neoadjuvant regimen in breast cancer patients.

Primary mediastinal large B-cell lymphoma: Clinical features, prognostic factors and survival with RCHOP in Arab patients in the PET scan era.

OBJECTIVE: PMBCL is a distinct type of nonhodgkins lymphoma with specific clinicopathological features. To clarify clinical features, treatment alternatives and outcomes, we evaluated 28 Arab patients treated with chemotherapy or radiotherapy between 2006 and 2011. PATIENTS AND METHODS: PMBCL lymphoma patients identified according to WHO classification and treated at KCCC between 2006 and 2011 were included in this study. Demographic and clinical data are presented as means or medians. Overall survival was estimated using the Kaplan-Meier method. Survival rates were compared using the log-rank test. A P < 0.05 was considered significant. RESULTS: The median age of the patients was 31 years and the male to female ratio was 2:1. Majority of the patients (75%) presented with stage I/II disease. Most had features of local extension like pleural effusion (18%) and SVCO (39%). Only 11% of the patients had bone marrow involvement at presentation. 96% of the patients required biopsy from the mediastinal mass either by image guided core biopsy (75%) or by surgical biopsy. Most patients were treated by RCHOP and involved field radiotherapy. Patients with positive PET scan after RCHOP chemotherapy received salvage chemotherapy and BEAM autologous marrow transplant. The five year OS for the entire group was 85% while the PFS was 73%. Patients who had PET scan for response evaluation had better OS [P = 0.013] and PFS [P = 0.039] when compared with those patients who received only radiotherapy based on CT scan evaluation. CONCLUSION: PMBCL is a specific lymphoma entity seen in the young with good survival. The role of PET scan for response evaluation and the type of consolidation therapy needs to be further clarified.

Pathological responses and long-term outcome analysis after neoadjuvant chemotheraphy in breast cancer patients from Kuwait over a period of 15 years.

BACKGROUND AND OBJECTIVES: The attainment of pathological complete response (pCR) after neoadjuvant chemotherapy has been taken as a surrogate marker for disease-free survival and overall survival. This is however dependent on various other parameters such as stage, grade, and biologic markers. DESIGN AND SETTINGS: This is a retrospective study of 365 patients with histologically confirmed non-met.astatic breast cancer patients treated with neoadjuvant chemotherapy at the Kuwait Cancer Control Centre between 1998 and 2009. PATIENTS AND METHODS: A total of 365 breast cancer patients who had received neoadjuvant chemotherapy from 1998-2009 were analyzed for the relationship of pCR with hormone status, Her2 status, histopathological subtype. Survival analysis was also conducted. RESULTS: Hormone receptor (HR) negative tumors had a higher pCR as against HR positive tumors, and the highest pCR in our analysis of pathological subtypes were seen in the HR+, Her2neu + and HR-, Her2neu + group. In our study, we could make out the paradoxes that well differentiated, and HR positive tumors had a better survival in spite of having lower pCR. The luminal A subtype also had a better overall survival than the triple negative subtype in spite of having lower pCR with neoadjuvant chemotherapy. CONCLUSION: Though the achievement of pCR retains its significance, it is more prognostic in HR negative tumors. The importance of HR receptor status, grade, and histopathological subtype in the long-term survival has been emphasized.

Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency.

Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5' sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.

Mechanisms underlying the protein-kinase mediated regulation of the HERG potassium channel synthesis.

The HERG (human ether-a-go-go related gene) potassium channel aids in the repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cyclic-AMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo.

The use of Bcl-2 over-expression to stabilize hybridomas specific to the HERG potassium channel.

We encountered a high degree of clonal hybridoma loss in the course of generating antibodies specific for the hERG potassium channel. A protein that is crucial for controlling heart rhythm, is abundant in parts of the brain and is abnormally expressed in some tumors. Intracellular domains of the protein were used for immunogens and generated adequate antibody responses in mice. Subsequent hybridomas created using Ag8 myeloma fusion partner yielded clones that secreted specific antibody but none could be successfully maintained in culture. A variety of mechanisms, including polyploidy inherent to hybridoma development or production of cytotoxic antibodies, may be responsible for eventual loss of cell viability by mechanisms that may include apoptosis. When spleen cells were fused to the NSO myeloma cell line that stably over-expresses the anti-apoptotic protein Bcl-2, hybridoma clones were generated that remained viable in culture with high level of hERG-specific antibody production. When the parental NSO cell line not over-expressing Bcl-2 was used, no stable hybridomas were produced. Antibodies secreted by NSO-Bcl-2 hybridomas were specific for hERG and performed well in immunoblot, immunoprecipitation and immunofluorescence assays. This work demonstrates a feasible option when faced with antigens that seem to be associated with clonal instability in the process of generating monoclonal antibodies.

Partially dominant mutant channel defect corresponding with intermediate LQT2 phenotype.

BACKGROUND: The hereditary Long QT Syndrome is a common cardiac disorder where ventricular repolarization is delayed, abnormally prolonging the QTc interval on electrocardiograms. LQTS is linked to various genetic loci, including the KCNH2 (HERG) gene that encodes the α-subunit of the cardiac potassium channel that carries I(Kr). Here, we report and characterize a novel pathologic missense mutation, G816V HERG, in a patient with sudden cardiac death. METHODS: Autopsy-derived tissue sample was used for DNA extraction and sequencing from an unexpected sudden death victim. The G816V HERG mutation was studied using heterologous expression in mammalian cell culture, whole cell patch clamp, confocal immunofluorescence, and immunochemical analyses. RESULTS: The mutant G816V HERG channel has reduced protein expression and shows a trafficking defective phenotype that is incapable of carrying current when expressed at physiological temperatures. The mutant channel showed reduced cell surface localization compared to wild-type HERG (WT HERG) but the mutant and wild-type subunits are capable of interacting. Expression studies at reduced temperatures enabled partial rescue of the trafficking defect with appearance of potassium currents, albeit with reduced current density and altered voltage-dependent activation. Lastly, we examined a potential role for hypokalemia as a contributory factor to the patient's lethal arrhythmia by possible low-potassium-induced degradation of WT HERG and haplo-insufficiency of G816V HERG. CONCLUSION: The G816V mutation in HERG causes a trafficking defect that acts in a partially dominant negative manner. This intermediate severity defect agrees with the mild clinical presentation in other family members harboring the same mutation. Possible hypokalemia in the proband induced WT HERG degradation combined with haplo-insufficiency may have further compromised repolarization reserve and contributed to the lethal arrhythmia.

PKA phosphorylation of HERG protein regulates the rate of channel synthesis.

Acute changes in cAMP and protein kinase A (PKA) signaling can regulate ion channel protein activities such as gating. Effects on channels due to chronic PKA signaling, as in stress or disease states, are less understood. We examined the effects of prolonged PKA activity on the human ether-a-go-go-related gene (HERG) K(+) channel in stably transfected human embryonic kidney (HEK)293 cells. Sustained elevation of cAMP by either chlorophenylthiol (CPT)-cAMP or forskolin increased the HERG channel protein abundance two- to fourfold within 24 h, with measurable difference as early as 4 h. The cAMP-induced augmentation was not due to changes in transcription and was specific for HERG compared with other cardiac K(+) channels (Kv1.4, Kv1.5, Kir2.1, and KvLQT1). PKA activity was necessary for the effect on HERG protein and did not involve other cAMP signaling pathways. Direct PKA phosphorylation of the HERG protein was responsible for the cAMP-induced augmentation. Enhanced abundance of HERG protein was detected in endoplasmic reticulum-enriched, Golgi, and plasma membrane without significant changes in trafficking rates or patterns. An increase in the K(+) current density carried by the HERG channel was also observed, but with a delay, suggesting that traffic to the surface is rate-limiting traffic. Acceleration of the HERG protein synthesis rate was the primary factor in the cAMP/PKA effect with lesser effects on protein stability. These results provide evidence for a novel mechanism whereby phosphorylation of a nascent protein dictates its rate of synthesis, resetting its steady-state abundance.

A-kinase anchoring protein targeting of protein kinase A and regulation of HERG channels.

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I(Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I(Kr) by cAMP.