Outcome and feasibility of radiotherapy bridging in large B-cell lymphoma patients receiving CD19 CAR T in the UK.

Radiotherapy (RT) has potential synergistic effects with chimeric antigen receptor (CAR) T but is not widely used as bridging therapy due to logistical challenges and lack of standardised protocols. We analysed RT bridging in a multicentre national cohort of large B-cell lymphoma patients approved for 3L axicabtagene ciloleucel or tisagenlecleucel across 12 UK centres. Of 763 approved patients, 722 were leukapheresed, 717 had data available on bridging therapy. 169/717 (24%) received RT bridging, 129 as single modality and 40 as combined modality treatment (CMT). Of 169 patients, 65.7% had advanced stage, 36.9% bulky disease, 86.5% elevated LDH, 41.7% international prognostic index (IPI) ≥3 and 15.2% double/triple hit at the time of approval. Use of RT bridging varied from 11% to 32% between centres and increased over time. Vein-to-vein time and infusion rate did not differ between bridging modalities. RT-bridged patients had favourable outcomes with 1-year progression-free survival (PFS) of 56% for single modality and 47% for CMT (1-year PFS 43% for systemic bridging). This is the largest cohort of LBCL patients receiving RT bridging prior to CAR T reported to date. Our results show that RT bridging can be safely and effectively used even in advanced stage and high-risk disease, with low dropout rates and excellent outcomes.

Improved outcomes of large B-cell lymphoma patients treated with CD19 CAR T in the UK over time.

The success of CD19 Chimeric antigen receptor (CAR) T-cell therapy in large B-cell lymphoma (LBCL) has been partially offset by toxicity and logistical challenges, which off-the-shelf agents like CD20xCD3 bispecific antibodies might potentially overcome. However, when using CAR T outcomes as the 'standard-of-care comparator̕ for relapsed/refractory (r/r) LBCL, a potential learning curve with implementing a novel, complex therapy like CAR T needs to be considered. To address this, we analysed 726 UK patients intended to be treated with CD19 CAR T for r/r LBCL and compared outcomes between the first year of the national CAR T programme (Era 1; 2019) and the more recent treatment era (Era 2; 2020-2022). We identified significant improvements for Era 2 versus Era 1 in dropout rate (17% vs. 27%, p = 0.001), progression-free survival (1-year PFS 50% vs. 32%, p < 0.001) and overall survival (1-year OS 60% vs. 40%, p < 0.001). We also observed increased use of bridging therapy, improvement in bridging outcomes, more tocilizumab/corticosteroid use, reduced high-grade cytokine release syndrome (4% vs. 9%, p = 0.01) and intensive care unit admissions (20% vs. 32%, p = 0.001). Our results demonstrate significant improvement in CAR T outcomes over time, highlighting the importance of using up-to-date clinical data when comparing CAR T against new treatment options for r/r LBCL.

Toxicity assessment of lead, nickel and cadmium on zebra fish augmented with Bacillus xiamenensis VITMSJ3: An insight on the defense mechanism against oxidative stress due to heavy metals.

Increased urbanization in recent years has let to discharge of heavy metals into the environment which has caused severe impacts on soil as well as water. Therefore the current study was aimed to assess the toxicity of lead (Pb), nickel (Ni), and cadmium (Cd) from the contaminated water using zebra fish Danio rerio and detoxification of metals upon augmentation with Bacillus xiamenensis. Exposure doses till 150 mg L-1 of Pb, Ni and Cd in water showed lethal effects on fish. Similarly the histopathological analysis showed severe tissue disruption in the gills and liver which were less upon supplementation with bacterial strain VITMSJ3. On the 20th day, the uptake concentration of Pb, Ni and Cd in zebra fish was found to be 87 mg L-1, 89 mg L-1 and 91 mg L-1 respectively with VITMSJ3, from the water. Antioxidant enzymatic activities showed an increase upon bacterial supplementation, which reduced the oxidative stress. Further SEM-EDAX analysis confirmed the presence of Pb, Ni and Cd ions adsorbed on the gills. The results clearly showed less oxidative damages in fish with increased head and reduced tail%. Overall, the results showed a significant difference (p < 0.05) among the treatments compared with the control.

CAR T in patients with large B-cell lymphoma not fit for autologous transplant.

Large B-cell lymphoma (LBCL) patients with comorbidities and/or advanced age are increasingly considered for treatment with CD19 CAR T, but data on the clinical benefit of CAR T in the less fit patient population are still limited. We analysed outcomes of consecutive patients approved for treatment with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) by the UK National CAR T Clinical Panel, according to fitness for autologous stem cell transplant (ASCT). 81/404 (20%) of approved patients were deemed unfit for ASCT. Unfit patients were more likely to receive tisa-cel versus axi-cel (52% vs. 48%) compared to 20% versus 80% in ASCT-fit patients; p < 0.0001. The drop-out rate from approval to infusion was significantly higher in the ASCT-unfit group (34.6% vs. 23.5%; p = 0.042). Among infused patients, response rate, progression-free and overall survival were similar in both cohorts. CAR T was well-tolerated in ASCT-unfit patients with an incidence of grade ≥3 cytokine release syndrome and neurotoxicity of 2% and 11%, respectively. Results from this multicentre real-world cohort demonstrate that CD19 CAR T can be safely delivered in carefully selected older patients and patients with comorbidities who are not deemed suitable for transplant.

A systematic review on the implementation of advanced and evolutionary biotechnological tools for efficient bioremediation of organophosphorus pesticides.

Ever since the concept of bioremediation was introduced, microorganisms, microbial enzymes and plants have been used as principal elements for Organophosphate pesticide (OPP) bioremediation. The enzyme systems and genetic profile of these microbes have been studied deeply in past years. Plant growth promoting rhizobacteria (PGPR) are considered as one of the potential candidates for OPP bioremediation and has been widely used to stimulate the phytoremediation potential of plants. Constructed wetlands (CWs) in OPP biodegradation have brought new prospects to microcosm and mesocosm based remediation strategies. Application of synthetic biology has provided a new dimension to the field of OPP bioremediation by introducing concepts like, gene manipulation andediting, expression and regulation of catabolic enzymes, implementation of whole-cell based and enzyme based biosensor systems for the detection and monitoring of OPP pollution in both terrestrial and aquatic environment. System biology and bioinformatics tools have rendered significant knowledge regarding the genetic, enzymatic and biochemical aspects of microbes and plants thereby, helping researchers to analyze the mechanism of OPP biodegradation. Structural biology has provided significant conceptual information regarding OPP biodegradation pathways, structural and functional characterization of metabolites and enzymes, enzyme-pollutant interactions, etc. Therefore, this review discussed the prospects and challenges of most advanced and high throughput strategies implemented for OPP biodegradation. The review also established a comparative analysis of various bioremediation techniques and highlighted the interdependency among them. The review highly suggested the simultaneous implementation of more than one remediation strategy or a combinational approach creating an advantageous hybrid technique for OPP bioremediation.

Staphylococcus xylosus VITURAJ10: Pyrrolo [1,2α] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) (PPDHMP) producing, potential probiotic strain with antibacterial and anticancer activity.

In the present study bacterial strain (VITURAJ10), isolated from goat milk was characterised for its probiotic potential. The various probiotic traits included tolerance to acidic pH (up to pH 3), bile salts (0.3%) and transit gut environment (simulated with digestive juices such as pepsin, Oxgall and pancreatin). The isolate could withstand high NaCl concentrations in the growth medium, showed inability to produce hemolysin and did not hydrolyse mucin. VITURAJ10 was capable of forming biofilm and produced exopolysachharide. The bioactive metabolites produced by the isolate were extracted and they showed growth suppressing activity towards pathogenic strains such as Escherichia coli, Salmonella enterica and Staphylococcus aureus. The crude extract was fractionated with solid phase extraction (SPE) chromatography and the fractions 10 and 12 were found to be effective against the bacterial pathogens. The fractions were further gauged for cytotoxic activity against MCF-7 cell line by MTT assay. The biologically significant compounds identified through GC-MS and FT-IR analysis in the fractions were, Actinomycin D, Pyrrolo [1,2α] Pyrazine-1,4-Dione, Hexahydro-3-(2-Methylpropyl)- (PPDHMP) and Didemnin B. The phylogenetic taxonomy of the isolate revealed the isolate to be the closest neighbour of Staphylococcus xylosus VITURAJ10 (GenBank accession no. KX770743.1) as per the16S rRNA gene sequencing and subsequent phylogenetic tree analysis.

Rapid biodegradation and biofilm-mediated bioremoval of organophosphorus pesticides using an indigenous Kosakonia oryzae strain -VITPSCQ3 in a Vertical-flow Packed Bed Biofilm Bioreactor.

The widespread use of pesticides has been one of the major anthropogenic sources of environmental pollution. Organophosphorus (OP) pesticides are predominantly used in agriculture due to their broad-spectrum insecticidal activity and chemical stability. The study was focused on the biodegradation of OP pesticides, Profenofos (PF) and Quinalphos (QP) in culture media using bacterium isolated from wetland paddy rhizosphere. The strain VITPSCQ3 showed higher pesticide tolerance, efficient biofilm formation and was capable of synthesizing organophosphate degrading enzymes. Based on the 16S rRNA gene sequencing the isolate exhibited maximum sequence similarity with Kosakinia oryzae (GenBank accession number: KR149275). Biodegradation assay with various concentrations of PF and QP (200, 400, 600 and 800 mg L-1) showed maximum degradation up to 82% and 92% within 48 h. The kinetic studies revealed the biodegradation rates (k) to be 0.0844 min-1 and 0.107 min-1 with half-lives (h) of 18 h and 14.8 h for PF and QP. The degradation products were identified by GCMS and possible degradation pathways were proposed using Insilico techniques. To the best of our knowledge, this is the first report on the biodegradation of PF and QP using Kosakonia oryzae. Bioremoval of PF and QP from aqueous solution was performed using the biofilm of VITPSCQ3 developed on selected substrates in a circulating Vertical-flow packed-bed biofilm (VFPBB) bioreactor. Charcoal, gravel and mushroom (Agaricus bisporus) were used as biofilm carriers. Mushroom showed strong biofilm formation with optimum biodegradation capacity of up to 96% for PF and 92% for QP within 120 min reaction time.

Reactive green dye remediation by Alternanthera philoxeroides in association with plant growth promoting Klebsiella sp. VITAJ23: A pot culture study.

Contamination of soil by textile effluent is a major threat found worldwide. These pollutants have diverse range of negative effects on the ecosystem, therefore restoration through cost effective biological strategy is the need of the hour. The aim of the current study was to enhance the decolourization of reactive green dye (RGD) using phytoremediation coupled with augmentation of effective bacteria to the rhizosphere. The isolate Klebsiella sp. VITAJ23 was isolated from textile effluent polluted soil and was assessed for its plant growth promoting traits (PGP) and the PGP functional genes were amplified. The soil was artificially polluted with RGD concentration ranging from 1000 to 3000 mg kg-1 and Alternanthera philoxeroides plantlets were planted in phyto and rhizoremediation treatments, the setup was maintained upto 60 d. The isolate VITAJ23 was augmented in the rhizoremediation setup and the morphological parameters were assessed at regular interval. There was a significant increase in the chlorophyll content as well as root and shoot length of the plant when treated with the bacterial suspension. Decolourization study revealed 79% removal of reactive green dye with an enhanced oxido-reductase enzyme activity in the setup bioaugmented with bacteria. The biodegraded metabolites were identified as 2-allylnapthalene, l-alanine, n-acetyl-and propenoic acid by GC-MS analysis and a plant-bacteria degradation pathway was predicted using computational tools. Inoculation of PGP-Klebsiella sp. VITAJ23 enhanced the rate of plant growth and dye degradation.

Enhanced uptake of Cd by biofilm forming Cd resistant plant growth promoting bacteria bioaugmented to the rhizosphere of Vetiveria zizanioides.

Heavy metals are the major cause of pollution and cadmium is one among the highly toxic metals discharged into the environment from various industries. The current study was focused on the bioremoval of cadmium by phyto and rhizoremediation approach using Vetiveria zizanioides. The bacterial strains were isolated from wetland paddy rhizosphere soil and the isolate VITJAN13 was found to be a biofilm forming Cd resistant plant growth promoting rhizobacteria (PGPR). The 16S rRNA gene sequencing revealed VITJAN13 to be the closest neighbor of Aeromonas sp. and was submitted to Genbank with the accession number KX770741. Further, pot culture studies indicated that the treatments bioaugmented with VITJAN13 increased the root length and shoot height by 21.4 and 17.36%, respectively as compared to the non-augmented plants. Hence, bioaugmentation of Aeromonas sp. in the rhizosphere of Vetiveria zizanioides enhanced the uptake of cadmium by 67.7% in the soil treated with 15 mg/kg of Cd to that of the phytoremediation setup.

Probiotic Bacteria in Wound Healing; An In-Vivo Study.

BACKGROUND: Probiotics are food supplements that benefit the host by improving its intestinal microbial balance. Probiotics are used as diet supplements to prevent diarrhoea and improve lactose tolerance. OBJECTIVES: The present study deals with the isolation of a potent probiotic strain capable of inducing healing properties in rat model. MATERIALS AND METHODS: Probiotic VITSAMJ1 was isolated from goat milk using MRS media. The antimicrobial assay was carried out against S. aureus (MTCC 3160) and the wound healing properties were assessed on female Wistar rats. A 1.5 cm2 subcutaneous wound was induced in the rats, and a probiotic gel formulation was tropically applied onto the wounds. Tissue biopsy was carried out after days 1, 3, 5, 7, 9, and 11. Total leucocyte count and Histopathological analysis were performed after each interval. RESULTS: VITSAMJ1 can be effectively used for wound healing. CONCLUSION: VITSAMJ1 can be effectively used for wound healing.

Fatal peripheral neuropathy following FLA chemotherapy.

We discuss a case with significant progressive peripheral neurological deterioration following administration of both fludarabine and cytarabine as part of the FLA (fludarabine and cytarabine) regime. Of particular interest is that toxicity only occurred during the second course of FLA and sixth course of Ara-C containing chemotherapy. At this point, a new antifungal agent had been commenced, suggesting a possible drug interaction enhancing the risk of known neurological toxicity with this regime.

Immediate haemostasis with recombinant factor VIIa for haemorrhage following Hickman line insertion in acute myeloid leukaemia.

Bleeding following Hickman line insertion is not uncommon but can be life threatening, especially in the presence of coagulopathy and thrombocytopenia following chemotherapy. Treatment to control the bleeding can be challenging and treatment options are limited. We present our experience of a patient who had persisting haemorrhage immediately following Hickman line insertion for administration of chemotherapy for relapsed acute myeloid leukaemia. Haemostasis could not be achieved after FFP and platelet administration. A single dose of recombinant factor VIIa (rhFVIIa) stopped the bleeding immediately, avoiding the need for surgical intervention or line removal. Our experience indicates rhFVIIa may be an effective option for bleeding related to Hickman line insertion.

Lentivirus administration to rat muscle provides efficient sustained expression of erythropoietin.

A lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding rat erythropoietin (EPO) complementary DNA was administered to rat skeletal muscle and red blood cell production was serially monitored. After a single intramuscular injection hematocrit values increased and reached a plateau at about 35 days and were sustained for at least 14 months. Virus doses of 6 x 10(7) infectious units and 6 x 10(6) infectious units produced significantly increased mean hematocrit values of 68.5% +/- 2.1% (P <.001, n = 4) and 52.7% +/- 1.3% (P <.001, n = 3), respectively, over values of control animals receiving normal saline (46.2% +/- 1.5%, n = 2). A polymerase chain reaction (PCR) assay for vector sequences in genomic DNA showed muscle tissue at the site of injection was positive and undetectable in liver, spleen, kidney, and lung. The intramuscular administration of lentivirus provided a dose-responsive, highly efficient and sustained EPO gene delivery, suggesting these vectors may be applied generally to the systemic delivery of proteins such as hormones and clotting factors. (Blood. 2001;98:594-596)

Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression.

Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Glucose-regulated insulin expression in diabetic rats.

Retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an amplified, glucose-regulated secretion of insulin. We constructed LhI*TFSN virus to encode a glucose-regulatable transforming growth factor alpha promoter controlling furin expression with a viral LTR promoter driving constitutive expression of furin-cleavable human proinsulin. Autologous BB rat vascular smooth muscle cells transduced with LhI*TFSN virus and cultured in 1.7 and 16.7 mM glucose secreted 50.7 +/- 3.2 and 136.0 +/- 11.0 microU (mean +/- SD) of insulin per 10(6) cells per day, respectively. After the onset of diabetes spontaneously diabetic congenic DR lyp/lyp BB rats received stomach implants containing 2 x 10(6) LhI*TFSN-transduced primary rat vascular smooth muscle cells. In eight treated rats there was a major reduction in insulin requirement to as low as 25% of pretreatment level for up to 3 months and one rat became insulin free without hypoglycemia. Intraperitoneal glucose tolerance tests (IPGTTs) in diabetic rats receiving control implants did not show the characteristic decline in blood glucose of normal rats after glucose administration. In contrast, diabetic rats receiving LhI*TFSN-transduced cells showed significant clearances of blood glucose. These data suggest clinically significant levels of glucose-regulated insulin delivery from implanted vascular smooth muscle cells transduced with LhI*TFSN vector.

Apical gene transfer into quiescent human and canine polarized intestinal epithelial cells by lentivirus vectors.

Intestinal epithelial cells secrete a protective luminal mucus barrier inhibiting viral gene transfer. Quiescent, polarized monolayers of primary epithelial cells from dog gallbladder and human colon are efficiently transduced through the apical mucus side by lentivirus vectors, suggesting their application to intestinal gene therapy.

Retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with viral particles and are devoid of viral vector RNA.

Obtaining high expression levels of a therapeutic gene in target cells could be achieved by integrating multiple copies of a recombinant retrovirus. However, we observed that cells retrovirally infected at high multiplicities of infection (MOIs) carried only single or double integrated proviral copies, suggesting that maximum retroviral transduction was achieved at relatively low MOIs. The same results were obtained when purified virus, free of most medium components, was used. Retroviral infection was shown to be inhibited by supernatants of other viral producer cell lines, and this inhibition could be removed by a centrifugation step that also removed more than 90% of infectious virus. Quantitative-competitive PCR of retroviral preparations showed that the amount of retroviral vector RNA present was similar to the amount expected on the basis of virus titers. Our data suggest that retroviral preparations derived from PA317 packaging cells contain inhibitors that copurify with retroviruses and do not contain viral vector RNA. We postulate that these inhibitor particles cannot achieve a productive infection but interfere with transduction of the target cells by infectious virions. This study might define an important criterion for the selection of more effective packaging cell lines.

Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand.

Efficient transduction of primary human T cells is an important goal toward treating a number of genetic defects. Patient T cells could be harvested by leukapheresis, transduced, and returned to the donor. A wide range of secreted or cell surface therapeutic proteins may be delivered in this way. The ability to produce antibodies is the consequence of interactions between T cells and B cells and lack of expression of CD40 ligand (CD40L) on T cells causes X-linked hyper-IgM syndrome (XHIM). We are investigating delivery of a normal CD40 ligand to treat this disorder. We tested promoters driving the expression of either reporter genes such as enhanced green fluorescent protein (eGFP) or human CDC40L. Using murine retroviruses, the best able to drive gene expression in T cells was the cytomegalovirus (CMV) promoter enhancer element; however, transduction efficiency was low. To achieve efficient, high-level gene expression we tested lentiviral gene delivery vectors. At a low multiplicity of infection (MOI) (0.5-2) a large fraction of target cells was transduced by lentiviral vectors (40-93%), and the strength of gene expression was high, as determined by flow cytometric analysis. We monitored the expression of eGFP or human CD40L on T cell lines and untransformed primary human T cells from normal and CD40L-deficient patients. We achieved efficient gene expression without an extended exposure to virus, and without the need for selection. These results are encouraging for efficient lentivirus-mediated transduction of refractory human cells to achieve therapeutic gene delivery.