Calvarial Lesions: A Tertiary Centre's Experience Over Fifteen Years.

BACKGROUND: The human calvaria harbors a variety of pathology and majority of them are incidentally noticed as painless swelling. The aim of the present study is to describe the histopathological subtypes of calvarial lesions, their management and factors affecting their surgical outcome at a tertiary care referral center. MATERIAL AND METHODS: All patients who underwent excision of the calvarial lesions over the last 15 years (from January 2005 to July 2019) were included in this study. Patients having calvarial pathology of infective origin and recurrent lesions were excluded. Any patient with multiple calvarial lesions who have been operated more than one time for same histopathological diagnosis was counted as one patient. We studied Karnofsky Performance Status (KPS) scores and radiological changes at 3-month follow up. RESULTS: Total 65 patients were recruited in this retrospective observational study. The median age of patients in the study was 29 years (range: 8 years to 68 years). Fibrous dysplasia 20 (30.7%) was the commonest lesion while metastatic thyroid carcinoma 3 (4.6%) was the most common malignant pathology. Complete excision was performed in 51 (78.5%) of patients while in 14 (21.5%) cases, subtotal or near total decompression were achieved. After three months of surgery, there was significant improvement in the KPS score (P < 0.00001). Duration of follow up ranges from 6 months to 5 years with 4 mortality in the study. CONCLUSIONS: Most of the calvarial tumors were benign and surgically addressable. The malignant lesions were scattered with diverse underlying pathology and required individualized holistic approach.

Efficacy of Silicone Conduit in the Rat Sciatic Nerve Repair Model: Journey of a Thousand Miles.

BACKGROUND: A lot of options have been tried for bridging the two ends of the injured nerves. Researchers have used decellularized nerve grafts, artificial materials and even nerve growth factors to augment functional recovery. These materials are either costly or inaccessible in developing world. OBJECTIVE: The study aimed to evaluate the efficacy of the silicone conduit in a rat sciatic nerve injury model. MATERIALS AND METHODS: 24 healthy Sprague-Dawley (SD) rats (250-300 grams; 8-10 weeks) were used and right sciatic nerve was exposed; transected and re-anastomosed by two different methods in 16 rats. In control group, n = 8 (Group I) the sciatic nerve was untouched; Group II (reverse nerve anastomosis, n = 8): 1-centimeter of nerve was cut and re-anastomosed by using 10-0 monofilament suture; Group III (silicone conduit, n = 8) 1-centimeter nerve segment was cut, replaced by silicone conduit and supplemented by fibrin glue]. Evaluation of nerve recovery was done functionally (pain threshold and sciatic functional index) over 3 months and histologically and electron microscopically. RESULTS: Functional results showed a trend of clinical improvement in Group III and II but recovery was poor and never reached up to normal. Histopathological and electron microscopic results showed an incomplete axonal regeneration in Groups II and III. Psychological analyses showed that no outwards signs of stress were present and none of the rats showed paw biting and teeth chattering. CONCLUSION: The silicone conduit graft may be an economical and effective alternative to presently available interposition grafts, however for short segments only.

Role of Essential Metal Ions in AAV Vector-Mediated Transduction.

Metal elements are essential components of approximately half of all cellular proteins, and approximately one-third of all known enzymes thus far are metalloenzymes. Several cellular proteins and enzymes undoubtedly impact the transduction efficiency of recombinant adeno-associated virus (AAV) vectors, but the precise role of metal ions in this process has not been studied in detail. In the present studies, we systematically evaluated the effects of all 10 essential metal ions (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc) on the transduction efficiency of AAV vectors. We report herein that five essential metal ions (iron, magnesium, manganese, molybdenum, and sodium) had little to no effect, and calcium strongly inhibited the transduction efficiency of AAV2 vectors. Whereas copper and potassium increased the transduction efficiency by ∼5-fold and ∼2-fold, respectively, at low concentrations, both essential metals were strongly inhibitory at higher concentrations. Calcium also inhibited the transduction efficiency by ∼3-fold. Two metal ions (cobalt and zinc) increased the transduction efficiency up to ∼10-fold in a dose-dependent manner. The combined use of cobalt and zinc resulted in more than an additive effect on AAV2 vector transduction efficiency (∼30-fold). The transduction efficiency of AAV serotypes 1 through 6 (AAV1-AAV6) vectors was also augmented by zinc. Similarly, the transduction of both single-stranded (ss) and self-complementary (sc) AAV3 vectors was enhanced by zinc. Zinc treatment also led to a dose-dependent increase in expression of a therapeutic protein, the human clotting factor IX (hF.IX), mediated by scAAV3 vectors in a human hepatic cell line. This simple strategy of essential metal ion-mediated enhancement may be useful to lower the dose of AAV vectors for their optimal use in human gene therapy.

Chinese Medicine Protein and Peptide in Gene and Cell Therapy.

The success of gene and cell therapy in clinic during the past two decades as well as our expanding ability to manipulate these biomaterials are leading to new therapeutic options for a wide range of inherited and acquired diseases. Combining conventional therapies with this emerging field is a promising strategy to treat those previously-thought untreatable diseases. Traditional Chinese medicine (TCM) has evolved for thousands of years in China and still plays an important role in human health. As part of the active ingredients of TCM, proteins and peptides have attracted long-term enthusiasm of researchers. More recently, they have been utilized in gene and cell therapy, resulting in promising novel strategies to treat both cancer and non-cancer diseases. This manuscript presents a critical review on this field, accompanied with perspectives on the challenges and new directions for future research in this emerging frontier.

Review of bioaerosols in indoor environment with special reference to sampling, analysis and control mechanisms.

Several tiny organisms of various size ranges present in air are called airborne particles or bioaerosol which mainly includes live or dead fungi and bacteria, their secondary metabolites, viruses, pollens, etc. which have been related to health issues of human beings and other life stocks. Bio-terror attacks in 2001 as well as pandemic outbreak of flue due to influenza A H1N1 virus in 2009 have alarmed us about the importance of bioaerosol research. Hence characterization i.e. identification and quantification of different airborne microorganisms in various indoor environments is necessary to identify the associated risks and to establish exposure threshold. Along with the bioaerosol sampling and their analytical techniques, various literatures revealing the concentration levels of bioaerosol have been mentioned in this review thereby contributing to the knowledge of identification and quantification of bioaerosols and their different constituents in various indoor environments (both occupational and non-occupational sections). Apart from recognition of bioaerosol, developments of their control mechanisms also play an important role. Hence several control methods have also been briefly reviewed. However, several individual levels of efforts such as periodic cleaning operations, maintenance activities and proper ventilation system also serve in their best way to improve indoor air quality.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions.

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2>AAV3=AAVLK03>AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤35, 36-50, and ≥51 years old). People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo.

OBJECTIVE: Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted. METHODS: We synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors. RESULTS: The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model. CONCLUSION: Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo.

OBJECTIVE: In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS: Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS: We observed that treatment with pristimerin, at as low as 1 µmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION: These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Lignin nanotubes as vehicles for gene delivery into human cells.

Lignin nanotubes (LNTs) synthesized from the aromatic plant cell wall polymer lignin in a sacrificial alumina membrane template have as useful features their flexibility, ease of functionalization due to the availability of many functional groups, label-free detection by autofluorescence, and customizable optical properties. In this report we show that the physicochemical properties of LNTs can be varied over a wide range to match requirements for specific applications by using lignin with different subunit composition, a function of plant species and genotype, and by choosing the lignin isolation method (thioglycolic acid, phosphoric acid, sulfuric acid (Klason), sodium hydroxide lignin), which influences the size and reactivity of the lignin fragments. Cytotoxicity studies with human HeLa cells showed that concentrations of up to 90 mg/mL are tolerated, which is a 10-fold higher concentration than observed for single- or multiwalled carbon nanotubes (CNTs). Confocal microscopy imaging revealed that all LNT formulations enter HeLa cells without auxiliary agents and that LNTs made from NaOH-lignin penetrate the cell nucleus. We further show that DNA can adsorb to LNTs. Consequently, exposure of HeLa cells to LNTs coated with DNA encoding the green fluorescent protein (GFP) leads to transfection and expression of GFP. The highest transfection efficiency was obtained with LNTs made from NaOH-lignin due to a combination of high DNA binding capacity and DNA delivery directly into the nucleus. These combined features of LNTs make LNTs attractive as smart delivery vehicles of DNA without the cytotoxicity associated with CNTs or the immunogenicity of viral vectors.

High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression.

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

A combined therapeutic approach for pyruvate dehydrogenase deficiency using self-complementary adeno-associated virus serotype-specific vectors and dichloroacetate.

We determined the ability of self-complementary adeno-associated virus (scAAV) vectors to deliver and express the pyruvate dehydrogenase E1alpha subunit gene (PDHA1) in primary cultures of skin fibroblasts from 3 patients with defined mutations in PHDA1 and 3 healthy subjects. Cells were transduced with scAAV vectors containing the cytomegalovirus promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a vector:cell ratio of 200. Transgene expression was measured 72h later. The transduction efficiency of scAAV2 and scAAV6 vectors was 3- to 5-fold higher than that of the other serotypes, which were subsequently used to transduce fibroblasts with wild-type PDHA1 cDNA under the control of the chicken beta-action (CBA) promoter at a vector:cell ratio of 1000. Total PDH-specific activity and E1alpha protein expression were determined 10 days post-transduction. Both vectors increased E1alpha expression 40-60% in both control and patient cells, and increased PDH activity in two patient cell lines. We also used dichloroacetate (DCA) to maximally activate PDH through dephosphorylation of E1alpha. Exposure for 24h to 5mM DCA increased PDH activity in non-transduced control (mean 37% increase) and PDH deficient (mean 44% increase) cells. Exposure of transduced patient fibroblasts to DCA increased PDH activity up to 90% of the activity measured in untreated control cells. DCA also increased expression of E1alpha protein and, to variable extents, that of other components of the PDH complex in both non-transduced and transduced cells. These data suggest that a combined gene delivery and pharmacological approach may hold promise for the treatment of PDH deficiency.

A dual role of EGFR protein tyrosine kinase signaling in ubiquitination of AAV2 capsids and viral second-strand DNA synthesis.

A 52 kd cellular protein, FK506-binding protein (FKBP52), phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), inhibits adeno-associated virus 2 (AAV2) second-strand DNA synthesis and transgene expression. FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and TC-PTP over-expression leads to improved viral second-strand DNA synthesis and improved transgene expression. In these studies, we observed that perturbation of EGFR-PTK signaling by a specific inhibitor, Tyrphostin 23 (Tyr23), augmented the transduction efficiency of the single-stranded AAV (ssAAV) vector as well as the self-complementary AAV (scAAV) vector. Similarly, tyrosine-dephosphorylation of FKBP52 by TC-PTP resulted in increased transduction by both vectors. These data suggested that EGFR-PTK signaling also affects aspects of AAV transduction other than viral second-strand DNA synthesis. We document that inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsids which, in turn, facilitates nuclear transport by limiting proteasome-mediated degradation of AAV vectors. We also document that Tyr23-mediated increase in AAV2 transduction efficiency is not further enhanced by a specific proteasome inhibitor, MG132. Thus, EGFR-PTK signaling modulates ubiquitin (Ub)/proteasome pathway-mediated intracellular trafficking as well as FKBP52-mediated second-strand DNA synthesis of AAV2 vectors. This has implications in the optimal use of AAV vectors in gene therapy.

Down-regulation of expression of rat pyruvate dehydrogenase E1alpha gene by self-complementary adeno-associated virus-mediated small interfering RNA delivery.

Mutations in the E1alpha subunit gene (PDHA1) of the pyruvate dehydrogenase complex (PDC) are common causes of congenital lactic acidosis. An animal model of E1alpha deficiency could provide insight into the pathological consequences of mutations and serve to test potential therapies. Small interfering RNAs (siRNAs) were designed to cleave the messenger RNA (mRNA) of the E1alpha subunit and were tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co-transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1alpha mRNA. Quantitative PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24h and up to 65% by 56h, compared to negative and positive controls. Since oligonucleotide-mediated siRNA delivery provided only transient suppression, we next selected two siRNA candidates and generated self-complementary, double-stranded adeno-associated virus (scAAV) vectors (serotypes 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72h after infection and 54-70% 10 days after infection in RLF cultures. The expression of E1alpha and the specific activity of pyruvate dehydrogenase were also decreased at 10 days after infection in RLF cultures. Thus, scAAV siRNA-mediated knockdown of PDHA1 gene expression provides a strategy that may be applied to create a useful animal model of PDC deficiency.

Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo.

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.

Heat-shock treatment-mediated increase in transduction by recombinant adeno-associated virus 2 vectors is independent of the cellular heat-shock protein 90.

Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression. These studies have implications in the optimal use of recombinant AAV vectors in human gene therapy.