Unconventional mechanism of action and resistance to rapalogs in renal cancer.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.

Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1.

Programmed death-ligand 1 (PD-L1) drives inhibition of antigen-specific T cell responses through engagement of its receptor programmed death-1 (PD-1) on activated T cells. Overexpression of these immune checkpoint proteins in the tumor microenvironment has motivated the design of targeted antibodies that disrupt this interaction. Despite clinical success of these antibodies, response rates remain low, necessitating novel approaches to enhance performance. Here, we report the development of antibody fusion proteins that block immune checkpoint pathways through a distinct mechanism targeting molecular trafficking. By engaging multiple receptor epitopes on PD-L1, our engineered multiparatopic antibodies induce rapid clustering, internalization, and degradation in an epitope- and topology-dependent manner. The complementary mechanisms of ligand blockade and receptor downregulation led to more durable immune cell activation and dramatically reduced PD-L1 availability in mouse tumors. Collectively, these multiparatopic antibodies offer mechanistic insight into immune checkpoint protein trafficking and how it may be manipulated to reprogram immune outcomes.

CD38-Specific Gallium-68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET.

The limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, a first-in-class peptide-based radiotracer, [68Ga]Ga-AJ206, is developed that can be seamlessly integrated into the standard clinical workflow and is specifically designed to noninvasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). A bicyclic peptide, AJ206, is synthesized and exhibits high affinity to CD38 (KD: 19.1 ± 0.99 × 10-9 m) by surface plasmon resonance. Further, [68Ga]Ga-AJ206-PET shows high contrast within 60 min and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detects CD38 expression in cell line-derived xenografts, patient-derived xenografts (PDXs), and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantifies CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following all-trans retinoic acid (ATRA) therapy. In conclusion, [68Ga]Ga-AJ206 exhibits the salient features required for clinical translation, providing CD38-specific high-contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Non-invasive PD-L1 quantification using [18F]DK222-PET imaging in cancer immunotherapy.

BACKGROUND: Combination therapies that aim to improve the clinical efficacy to immune checkpoint inhibitors have led to the need for non-invasive and early pharmacodynamic biomarkers. Positron emission tomography (PET) is a promising non-invasive approach to monitoring target dynamics, and programmed death-ligand 1 (PD-L1) expression is a central component in cancer immunotherapy strategies. [18F]DK222, a peptide-based PD-L1 imaging agent, was investigated in this study using humanized mouse models to explore the relationship between PD-L1 expression and therapy-induced changes in cancer. METHODS: Cell lines and xenografts derived from three non-small cell lung cancers (NSCLCs) and three urothelial carcinomas (UCs) were used to validate the specificity of [18F]DK222 for PD-L1. PET was used to quantify anti-programmed cell death protein-1 (PD-1) therapy-induced changes in PD-L1 expression in tumors with and without microsatellite instability (MSI) in humanized mice. Furthermore, [18F]DK222-PET was used to validate PD-L1 pharmacodynamics in the context of monotherapy and combination immunotherapy in humanized mice bearing A375 melanoma xenografts. PET measures of PD-L1 expression were used to establish a relationship between pathological and immunological changes. Lastly, spatial distribution analysis of [18F]DK222-PET was developed to assess the effects of different immunotherapy regimens on tumor heterogeneity. RESULTS: [18F]DK222-PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high but variable tumor uptake at 60 min that correlated with PD-L1 expression. In MSI tumors treated with anti-PD-1, [18F]DK222 uptake was higher than in control tumors. Moreover, [18F]DK222 uptake was higher in A375 tumors treated with combination therapy compared with monotherapy, and negatively correlated with final tumor volumes. In addition, a higher number of PD-L1+ cells and higher CD8+-to-CD4+ cell ratio was observed with combination therapy compared with monotherapy, and positively correlated with PET. Furthermore, spatial distribution analysis showed higher [18F]DK222 uptake towards the core of the tumors in combination therapy, indicating a more robust and distinct pattern of immune cell infiltration. CONCLUSION: [18F]DK222-PET has potential as a non-invasive tool for monitoring the effects of immunotherapy on tumors. It was able to detect variable PD-L1 expression in tumors of different cancer types and quantify therapy-induced changes in tumors. Moreover, [18F]DK222-PET was able to differentiate the impact of different therapies on tumors.

PET/MRI and Bioluminescent Imaging Identify Hypoxia as a Cause of Programmed Cell Death Ligand 1 Image Heterogeneity.

Purpose To examine the association between hypoxia and programmed cell death ligand 1 (PD-L1) expression using bioluminescence imaging (BLI) and PET/MRI in a syngeneic mouse model of triple-negative breast cancer (TNBC). Materials and Methods PET/MRI and optical imaging were used to determine the role of hypoxia in altering PD-L1 expression using a syngeneic TNBC model engineered to express luciferase under hypoxia. Results Imaging showed a close spatial association between areas of hypoxia and increased PD-L1 expression in the syngeneic murine (4T1) tumor model. Mouse and human TNBC cells exposed to hypoxia exhibited a significant increase in PD-L1 expression, consistent with the in vivo imaging data. The role of hypoxia in increasing PD-L1 expression was further confirmed by using The Cancer Genome Atlas analyses of different human TNBCs. Conclusion These results have identified the potential role of hypoxia in contributing to PD-L1 heterogeneity in tumors by increasing cancer cell PD-L1 expression. Keywords: Hypoxia, PD-L1, Triple-Negative Breast Cancer, PET/MRI, Bioluminescence Imaging Supplemental material is available for this article. © RSNA, 2023.

A Gallium-68-Labeled Peptide Radiotracer For CD38-Targeted Imaging In Multiple Myeloma With PET.

PURPOSE: The limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, we developed [68Ga]Ga-AJ206, a peptide-based radiotracer that can be seamlessly integrated into the standard clinical workflow and is specifically designed to non-invasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). EXPERIMENTAL DESIGN: We synthesized a high-affinity binder for quantification of CD38 levels. Affinity was tested using surface plasmon resonance, and In vitro specificity was evaluated using a gallium-68-labeled analog. Distribution, pharmacokinetics, and CD38 specificity of the radiotracer were assessed in MM cell lines and in primary patient-derived myeloma cells and xenografts (PDX) with cross-validation by flow cytometry and immunohistochemistry. Furthermore, we investigated the radiotracer's potential to quantify CD38 pharmacodynamics induced by all-trans retinoic acid therapy (ATRA). RESULTS: [68Ga]Ga-AJ206 exhibited high CD38 binding specificity (KD: 19.1±0.99 nM) and CD38-dependent In vitro binding. [68Ga]Ga-AJ206-PET showed high contrast within 60 minutes and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detected CD38 expression in xenografts, PDXs and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantified CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following ATRA therapy. CONCLUSIONS: [68Ga]Ga-AJ206 exhibited the salient features required for clinical translation, providing CD38-specific high contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement.

Gallium-68-labeled Peptide PET Quantifies Tumor Exposure of PD-L1 Therapeutics.

PURPOSE: Immune checkpoint therapy (ICT) is currently ineffective in a majority of patients. Tumor drug exposure measurements can provide vital insights into mechanisms involved in the resistance of solid tumors to those therapeutics; however, tools to quantify in situ drug exposure are few. We have investigated the potential of programmed death-ligand 1 (PD-L1) pharmacodynamics, quantified using PET, to inform on the tumor exposure of anti-PD-L1 (aPD-L1) therapeutics. EXPERIMENTAL DESIGN: To noninvasively quantify PD-L1 levels, we first developed a novel peptide-based gallium-68-labeled binder, [68Ga]Ga-DK223, and evaluated its in vivo distribution, pharmacokinetics, and PD-L1 specificity in preclinical models of triple-negative breast cancer and urothelial carcinoma with variable PD-L1 expression. We then quantified baseline and accessible PD-L1 levels in tumors as a noninvasive pharmacodynamic measure to assess tumor exposure to two aPD-L1 antibodies (avelumab and durvalumab). RESULTS: DK223 exhibited a KD of 1.01±0.83 nmol/L for PD-L1 and inhibited the PD-1:PD-L1 interaction in a dose-dependent manner. [68Ga]Ga-DK223 provides high-contrast PET images within 60 minutes of administration and detects PD-L1 in an expression-dependent manner in xenograft models. PD-L1 pharmacodynamics measured using [68Ga]Ga-DK223-PET revealed that avelumab and durvalumab had similar exposure early during therapy, but only durvalumab exhibited sustained exposure at the tumor. CONCLUSIONS: [68Ga]Ga-DK223 detected variable PD-L1 levels and exhibited salient features required for clinical translation. [68Ga]Ga-DK223-PET could be useful for quantifying total PD-L1 levels at baseline and accessible PD-L1 levels during therapy to understand drug exposure at the tumor, thus supporting its use for guiding and optimizing ICT.

Immunogenicity of two COVID-19 vaccines used in India: An observational cohort study in health care workers from a tertiary care hospital.

COVID-19 pandemic witnessed rapid development and use of several vaccines. In India, a country-wide immunization was initiated in January 2021. COVISHIELD, the chimpanzee adenoviral-vectored vaccine with full-length SARS-COV-2 spike insert and COVAXIN, the whole virus-inactivated vaccines were used. To assess and compare immune response of health-care-workers to COVISHIELD (n=187) and COVAXIN (n=21), blood samples were collected pre-vaccination, 1month post-1/post-2 doses and 6months post-dose-2 and tested for IgG-anti-SARS-CoV-2 (ELISA) and neutralizing (Nab,PRNT50) antibodies. Spike-protein-specific T cells were quantitated by IFN-γ-ELISPOT. In pre-vaccination-antibody-negative COVISHIELD recipients (pre-negatives, n=120), %Nab seroconversion (median, IQR Nab titers) increased from 55.1% (16, 2.5-36.3) post-dose-1 to 95.6% (64.5, 4.5-154.2, p<0.001) post-dose-2 that were independent of age/gender/BMI. Nab response was higher among pre-positives with hybrid immunity at all-time points (p<0.01-0.0001) and independent of age/gender/BMI/Comorbidities. Post-dose-2-seroconversion (50%, p<0.001) and Nab titers (6.75, 2.5-24.8, p<0.001) in COVAXIN-recipients were lower than COVISHIELD. COVAXIN elicited a superior IFN-γ-T cell response as measured by ELISPOT (100%; 1226, 811-1532 spot forming units, SFU/million PBMCs v/s 57.8%; 21.7,1.6-169.2; p<0.001). At 6months, 28.3% (15/53) COVISHIELD and 3/3COVAXIN recipients were Nab-negative. T cell response remained unchanged. During immunization, COVID-19 cases were detected among COVISHIELD (n=4) and COVAXIN (n=2) recipients. At 6months, 9cases were recorded in COVISHIELD-recipients. This first-time, systematic, real-world assessment and long-term follow up revealed generation of higher neutralizing antibody titers by COVISHIELD and stronger T-cell response by COVAXIN. Diminished Nab titers at 6months emphasize early booster. Immunogenicity/efficacy of vaccines will change with the progression of the pandemic needing careful evaluations in the field-settings.

HIF2 Inactivation and Tumor Suppression with a Tumor-Directed RNA-Silencing Drug in Mice and Humans.

PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that ∼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.

Metasurfaces for Sensing Applications: Gas, Bio and Chemical.

Performance of photonic devices critically depends upon their efficiency on controlling the flow of light therein. In the recent past, the implementation of plasmonics, two-dimensional (2D) materials and metamaterials for enhanced light-matter interaction (through concepts such as sub-wavelength light confinement and dynamic wavefront shape manipulation) led to diverse applications belonging to spectroscopy, imaging and optical sensing etc. While 2D materials such as graphene, MoS2 etc., are still being explored in optical sensing in last few years, the application of plasmonics and metamaterials is limited owing to the involvement of noble metals having a constant electron density. The capability of competently controlling the electron density of noble metals is very limited. Further, due to absorption characteristics of metals, the plasmonic and metamaterial devices suffer from large optical loss. Hence, the photonic devices (sensors, in particular) require that an efficient dynamic control of light at nanoscale through field (electric or optical) variation using substitute low-loss materials. One such option may be plasmonic metasurfaces. Metasurfaces are arrays of optical antenna-like anisotropic structures (sub-wavelength size), which are designated to control the amplitude and phase of reflected, scattered and transmitted components of incident light radiation. The present review put forth recent development on metamaterial and metastructure-based various sensors.

Molecular Genetic Determinants of Shorter Time on Active Surveillance in a Prospective Phase 2 Clinical Trial in Metastatic Renal Cell Carcinoma.

Active surveillance (AS) may be used in the management of metastatic renal cell carcinoma (mRCC), but consensus regarding its application is lacking. We report an exploratory analysis of prospectively collected specimens prespecified in the only modern clinical trial evaluating AS in mRCC. Whole-exome and RNA sequencing were performed for patients providing consent to identify putative biomarkers associated with time on AS (TAS), the primary endpoint. Log-rank tests and multivariable Cox proportional-hazards models were used for analyses. Patients with mutations in either TP53 or SMARCA4 tumor suppressor genes had shorter TAS (7.5 vs 14.2 mo; log-rank p = 0.004). While these patients exhibited features of aggressive disease clinically, the two-gene model was independently predictive in multivariable analyses (hazard ratio 3.30, 95% confidence interval 1.07-10.18; p = 0.038). In conclusion, insight into the underlying RCC biology improves patient selection for AS. If validated, this two-gene model could help in stratifying patients with mRCC and identifying those who are poor candidates for AS. PATIENT SUMMARY: In this study, we analyzed tumors from patients with metastatic kidney cancer enrolled in a clinical trial of imaging surveillance. We found that tumors with mutations in either the TP53 or SMARCA4 gene progressed faster than tumors without these mutations. Thus, patients harboring mutations in these genes may not be good candidates for AS.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes.

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5'→3', 3' →5' or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically.Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm.

Doxorubicin-loaded MePEG-PCL nanoparticles for prevention of posterior capsular opacification.

AIMS: Cytotoxic drugs are considered as potent candidates for the prevention of posterior capsular opacification (PCO), but the toxicity incited to healthy intraocular structures is a major concern. In this study, the authors evaluated the effect of PEG methyl ether-block-poly(ε-caprolactone) (MePEG-PCL) doxorubicin (DOX)-loaded nanoparticles (NPs) for prevention of PCO and their influence on intraocular tissues. METHODS: MePEG-PCL DOX NPs were prepared and characterized. The cytotoxic effect of DOX NPs on lens epithelial cells was compared with free drug. Its effect on PCO prevention following single subconjunctival delivery to lensectomized rabbits was assessed. Toxicity to intraocular structures was evaluated by specular microscopy, electroretinography and histopathology. The availability of DOX in aqueous humor was determined by HPLC. RESULTS: The cytotoxic effect of DOX NPs was higher compared with free DOX due to prolonged retention within the cells. A significant reduction in degree of PCO was observed in DOX NP-treated eyes compared with untreated controls. There was no significant change in the density and morphology of corneal endothelial cells or the histology of intraocular structures. Electroretinographs of treated eyes did not change compared with the pretreatment values. DOX could be detected by HPLC in the aqueous humor up to 48 h following single subconjunctival injection. CONCLUSION: The authors conclude that DOX-loaded MePEG-PCL NPs show promise as a new approach to selectively kill highly proliferative lens epithelial cells in vivo following cataract surgery, while sparing normal tissue.

Modulation of matrix metalloproteinase activity by EDTA prevents posterior capsular opacification.

PURPOSE: To evaluate the effect of ethylenediaminetetraacetic acid (EDTA) on posterior capsular opacification (PCO) of rabbits and to assess its effect on intraocular tissues. METHODS: Modulation of matrix metalloproteinase (MMP) activity in the aqueous following cataract surgery in rabbits and its prevention by different doses of EDTA was determined by zymography. For evaluation of PCO, lensectomized rabbits were intracamerally injected with single dose of either 5 mg EDTA or normal saline. After one month, the degree of PCO was determined by slitlamp biomicroscopy, Miyake-Apple view, and histology of the lens capsule. The effect of EDTA on intra ocular pressure (IOP), corneal endothelial cells, and the retina was evaluated by tonometry, specular microscopy and scanning electron microscopy, and electroretinography. The concentration of EDTA in the aqueous was determined by high performance liquid chromatography (HPLC) at different time points. RESULTS: The MMP activity was significantly increased in the aqueous of the operated eyes, and EDTA reduced the degree of increase in a dose-dependent manner. EDTA treatment significantly reduced the degree of PCO (p<0.05). Histopathology of the lens capsule showed a reduction in the number of proliferating and migrating cells as well as MMP2 expression in the EDTA-treated eyes. EDTA treatment did not change the IOP; density, morphology and ultrastructure of the corneal endothelial cells; and electroretinography (ERG). EDTA was detectable in the aqueous humor up to 72 h following a single intracameral injection. CONCLUSIONS: EDTA reduces the degree of PCO by suppressing the MMP activity and it is not toxic to intra ocular structures at the concentration used.

Pathologic study of pandemic influenza A (H1N1) 2009 cases from India.

The pandemic influenza A (H1N1) 2009 originated in Mexico and rapidly spread to the United States and many other countries. India reported the first pandemic influenza case in May 2009. Autopsy studies describing the pathology of pandemic influenza infection in humans have appeared in the literature and most of these were from Western countries. We present the clinicopathologic features in 46 fatal cases with confirmed pandemic influenza A (H1N1) 2009 virus infection during August 2009 to October 2010. Postmortem needle biopsy tissues were examined for histopathological changes and distribution of virus antigen by immunohistochemistry. The results are comparable with previous autopsy studies. Diffuse alveolar damage was the consistent finding in the lung tissues. However, underlying medical conditions were not noted in the cases from present study. Consistent presence of viral antigen was noted in the bronchiolar epithelium without any reference to the duration of illness. This study also emphasizes the use of the postmortem needle biopsy technique whenever an autopsy is not possible.

Chandipura virus encephalitis outbreak among children in Nagpur division, Maharashtra, 2007.

BACKGROUND & OBJECTIVES: An outbreak of acute encephalitis syndrome (AES) among children from Nagpur division, Maharashtra was investigated to confirm the aetiology and to describe clinico-epidemiological features. METHODS: AES cases among children<15 yr, from Nagpur division, hospitalized between June-September 2007, were investigated. Serum and cerebrospinal fluid (CSF) were tested for IgM antibodies against Chandipura virus (CHPV) and Japanese encephalitis virus (JEV) and for CHPV RNA by RT-PCR. Partial N gene sequences were used for phylogenetic analysis. Virus isolations were attempted in rhabdomyosarcoma (RD) cell line. Sandflies were collected, pooled and tested for CHPV RNA by RT-PCR. RESULTS: A total of 78 AES cases were recorded in children<15 yr of age. Case fatality ratio was 43.6 per cent. Male to female ratio was 1:1.2. Chandipura (CHP) was confirmed in 39 cases. CHPV RNA was detected in both CSF and serum specimens of 2 cases and in serum of 22 cases. Phylogenetic analysis showed 99.98-100 per cent nucleotide identity in the sequences studied. Anti-CHPV IgM antibodies were detected in CSF of 2 cases and in serum of 8 cases. Seroconversion to anti-CHPV IgM antibodies was observed in 5 cases. Clinical manifestations of CHP cases (n=38) were fever (100%), convulsion (76.3%), altered sensorium (34.2%), headache (23.7%), vomiting (44.7%) and diarrhoea (23.7%). CHPV RNA was detected in one of two pools of sandflies from affected locality. INTERPRETATION & CONCLUSIONS: Chandipura virus was confirmed as the aetiological agent of this acute encephalitis outbreak with high case-fatality among children.

Host gene expression profiling in influenza A virus-infected lung epithelial (A549) cells: a comparative analysis between highly pathogenic and modified H5N1 viruses.

BACKGROUND: To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549) at early and late stages of infection with highly pathogenic avian Influenza A (H5N1) virus and compared it with a reverse genetics modified recombinant A (H5N1) vaccine virus using microarray platform. RESULTS: The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi). Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi. CONCLUSION: We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent.