Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines.

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Omental desmoplastic small round cell tumor with metastasis.

Desmoplastic small round cell tumor (DSRCT) is a very rare diagnosis with about 200 cases reported in literature. DSRCT is a recently described histopathological entity by Gerald and Rosai in 1989. Abdominopelvic cavity especially peritoneum is the most common site. We report a case of a huge omental DSRCT with lymph node metastasis which was initially misdiagnosed as gastrointestinal stromal tumor on radiology. A 26-year-old male presented with complaints of upper abdominal swelling associated with constant dull pain. On examination there was a large 15 × 12 cm intraabdominal mass in the epigastric and umbilical region. Imaging studies were suggestive of neoplastic mesenchymal etiology. Image-guided fine-needle aspiration cytology (FNAC) was suggestive of mesenchymal neoplastic etiology. On laparotomy, there was a huge 20 × 15 cm mass arising from omentum with multiple omental and mesenteric seedlings and mesenteric, peripancreatic and perigastric lymphadenopathy. The patient underwent debulking surgery with uneventful post-operative recovery. Histopathological examination with immunohistochemistry revealed a diagnosis of DSRCT of omentum and small bowel mesentery with lymph node metastasis. Patient then received adjuvant chemotherapy with multiple chemotherapeutic drugs as per P6 protocol and has stable disease at 1 year follow up.

A clinicopathological study of peripheral schwannomas.

AIM AND OBJECTIVE: Schwannomas are benign neoplasms of neural origin with sporadic or syndromic occurence. They are commonly seen in cranial nerves. Peripheral schwannomas occur rarely and may have unique presentations. The aim of this study is to evaluate the clinico-pathological characteristics of peripheral schwannomas. METHODS: A retrospective cross sectional study of peripheral schwannomas excluding head neck region was conducted. The study group consisted of 18 cases which were recorded over a period of seven years. The corresponding data were collected from the archives of the Department of Pathology. RESULTS: Male to female ratio was 1:1. The average age of the cases was 47 years. The most common site was the upper limbs (55.55%) followed by lower limbs, chest and penis. The lesions mostly presented as painless swellings (62%). Histopathological examination revealed classic features of schwannoma. Secondary changes included cystic degeneration, foam cells, epitheloid cells, hyalinization, microcystic change and collection of plasma cells. All cases were confirmed by positive S100 staining. CONCLUSION: Peripheral schwannomas may be missed due to its rarity and atypical presentations. Both clinicians and pathologists should be aware of this common entity at unusual sites for the proper management of the patients. Surgery is usually the treatment of choice.

Management of early melanoma recurrence despite adjuvant anti-PD-1 antibody therapy☆.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies (PD1) prolong recurrence-free survival in high-risk resected melanoma; however, approximately 25%-30% of patients recur within 1 year. This study describes the pattern of recurrence, management and outcomes of patients who recur with adjuvant PD1 therapy. PATIENTS AND METHODS: Consecutive patients from 16 centres who recurred having received adjuvant PD1 therapy for resected stage III/IV melanoma were studied. Recurrence characteristics, management and outcomes were examined; patients with mucosal melanoma were analysed separately. RESULTS: Melanoma recurrence occurred in 147 (17%) of ∼850 patients treated with adjuvant PD1. In those with cutaneous melanoma (n = 136), median time to recurrence was 4.6 months (range 0.3-35.7); 104 (76%) recurred during (ON) adjuvant PD1 after a median 3.2 months and 32 (24%) following (OFF) treatment cessation after a median 12.5 months, including in 21 (15%) who ceased early for toxicity. Fifty-nine (43%) recurred with locoregional disease only and 77 (57%) with distant disease. Of those who recurred locally, 22/59 (37%) subsequently recurred distantly. Eighty-nine (65%) patients received systemic therapy after recurrence. Of those who recurred ON adjuvant PD1, none (0/6) responded to PD1 alone; 8/33 assessable patients (24%) responded to ipilimumab (alone or in combination with PD1) and 18/23 (78%) responded to BRAF/MEK inhibitors. Of those who recurred OFF adjuvant PD1, two out of five (40%) responded to PD1 monotherapy, two out of five (40%) responded to ipilimumab-based therapy and 9/10 (90%) responded to BRAF/MEK inhibitors. CONCLUSIONS: Most patients who recur early despite adjuvant PD1 develop distant metastases. In those who recur ON adjuvant PD1, there is minimal activity of further PD1 monotherapy, but ipilimumab (alone or in combination with PD1) and BRAF/MEK inhibitors have clinical utility. Retreatment with PD1 may have activity in select patients who recur OFF PD1.

Current Status and Future Perspective for Research on Medicinal Plants with Anticancerous Activity and Minimum Cytotoxic Value.

Cancer is the second leading cause of morbidity and mortality worldwide. Although chemotherapy and radiotherapy enhance the survival rate of cancerous patients but they have several acute toxic effects. Therefore, there is a need to search for new anticancer agents having better efficacy and lesser side effects. In this regard, herbal treatment is found to be a safe method for treating and preventing cancer. Here, an attempt has been made to screen some less explored medicinal plants like Ammania baccifera, Asclepias curassavica, Azadarichta indica, Butea monosperma, Croton tiglium, Hedera nepalensis, Jatropha curcas, Momordica charantia, Moringa oleifera, Psidium guajava, etc. having potent anticancer activity with minimum cytotoxic value (IC50 >3µM) and lesser or negligible toxicity. They are rich in active phytochemicals with a wide range of drug targets. In this study, these medicinal plants were evaluated for dose-dependent cytotoxicological studies via in vitro MTT assay and in vivo tumor models along with some more plants which are reported to have IC50 value in the range of 0.019-0.528 mg/ml. The findings indicate that these plants inhibit tumor growth by their antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic molecular targets. They are widely used because of their easy availability, affordable price and having no or sometimes minimal side effects. This review provides a baseline for the discovery of anticancer drugs from medicinal plants having minimum cytotoxic value with minimal side effects and establishment of their analogues for the welfare of mankind.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

A novel agent SL-401 induces anti-myeloma activity by targeting plasmacytoid dendritic cells, osteoclastogenesis and cancer stem-like cells.

Novel therapies for multiple myeloma (MM) can target mechanism(s) in the host-MM bone marrow (BM) microenvironment mediating MM progression and chemoresistance. Our studies showed increased numbers of tumor-promoting, immunosuppressive and drug-resistant plasmacytoid dendritic cells (pDCs) in the MM BM microenvironment. pDC-MM cell interactions upregulate interleukin-3 (IL-3), which stimulates both pDC survival and MM cell growth. Since IL-3 R is highly expressed on pDCs in the MM BM milieu, we here targeted pDCs using a novel IL-3 R-targeted therapeutic SL-401. In both in vitro and in vivo models of MM in its BM milieu, SL-401 decreases viability of pDCs, blocks pDC-induced MM cell growth, and synergistically enhances anti-MM activity of bortezomib and pomalidomide. Besides promoting pDC survival and MM cell growth, IL-3 also mediates progression of osteolytic bone disease in MM. Osteoclast (OCL) progenitor cells express IL-3 R, and we show that SL-401 abrogates monocyte-derived OCL formation and bone resorption. Finally, we show that SL-401 also decreases the viability of IL-3 R-expressing cancer stem-like cells in MM. Overall, our study provides the preclinical basis for clinical trials of SL-401 to block pDC-induced MM cell growth, inhibit osteoclastogenesis and target MM stem-like cell subpopulations to improve patient outcome in MM.

Weekly versus Three-Weekly Cisplatin-based Concurrent Chemoradiotherapy as definitive treatment in Head and Neck Cancer- Where do we stand?

PURPOSE: To compare toxicity, compliance, and early response of weekly and 3-weekly cisplatin administration concurrent with radiotherapy as definitive treatment in locally advanced squamous cell carcinoma head and neck. MATERIALS AND METHODS: Patients with histologically proven stage III - IV B head and neck carcinoma presenting from June 2013 to March 2014 were randomly assigned to weekly (35 mg/m2, 6 cycles; arm A) and 3 weekly (100 mg/m2, 3 cycles; arm B) cisplatin with concurrent radiotherapy. RESULTS: 60 patients were randomly assigned to treatment, 30 in each arm. Median follow-up was 8 months (range 4-13). There was no significant difference in grade 3 mucositis between the two arms (75.9% vs 70%, p = 0.20). Grade 3 neutropenia was more frequent in arm B (55.2% vs 26.7%, p = 0.01). Hypomagnesemia was the commonest electrolyte imbalance and it was significantly higher in arm B (60% vs 20%, p = 0.001). Completion rate of scheduled chemotherapy cycles was higher for patients receiving weekly regimen. Response at 3 months was similar for all the patients {Complete Response (66.7% vs 62.1%), p = 0.200}. Our data suggested that there is a reduced need of hospitalization and supportive care measures for patients receiving weekly cisplatin with RT (p = 0.05). CONCLUSIONS: Weekly cisplatin 35 mg/m2 chemotherapy concurrent with radiotherapy is equally effective and less toxic in terms of neutropenia, hypomagnesemia and need for supportive measures than the conventional 3 weekly cisplatin 100 mg/m2 regimen.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells.

The hypoxic bone marrow (BM) microenvironment confers growth/survival and drug resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and nitric oxide-donating properties. Here, we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001-induced apoptosis is associated with: (i) activation of caspases; (ii) release of ROS and nitrogen species; (iii) induction of DNA damage via ATM/γ-H2AX; and (iv) decrease in DNA methyltransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. The deubiquitylating enzyme USP7 stimulates DNMT1 activity, and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.

Targeting proteasome ubiquitin receptor Rpn13 in multiple myeloma.

Proteasome inhibitor bortezomib is an effective therapy for relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance can limit its long-term utility. Recent research has focused on targeting ubiquitin receptors upstream of 20S proteasome, with the aim of generating less toxic therapies. Here we show that 19S proteasome-associated ubiquitin receptor Rpn13 is more highly expressed in MM cells than in normal plasma cells. Rpn13-siRNA (small interfering RNA) decreases MM cell viability. A novel agent RA190 targets Rpn13 and inhibits proteasome function, without blocking the proteasome activity or the 19S deubiquitylating activity. CRISPR/Cas9 Rpn13-knockout demonstrates that RA190-induced activity is dependent on Rpn13. RA190 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma and overcomes bortezomib resistance. Anti-MM activity of RA190 is associated with induction of caspase-dependent apoptosis and unfolded protein response-related apoptosis. MM xenograft model studies show that RA190 is well tolerated, inhibits tumor growth and prolongs survival. Combining RA190 with bortezomib, lenalidomide or pomalidomide induces synergistic anti-MM activity. Our preclinical data validates targeting Rpn13 to overcome bortezomib resistance, and provides the framework for clinical evaluation of Rpn13 inhibitors, alone or in combination, to improve patient outcome in MM.

A novel TLR-9 agonist C792 inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib.

Our prior study in multiple myeloma (MM) patients showed increased numbers of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM), which both contribute to immune dysfunction as well as promote tumor cell growth, survival and drug resistance. Here we show that a novel Toll-like receptor (TLR-9) agonist C792 restores the ability of MM patient-pDCs to stimulate T-cell proliferation. Coculture of pDCs with MM cells induces MM cell growth; and importantly, C792 inhibits pDC-induced MM cell growth and triggers apoptosis. In contrast, treatment of either MM cells or pDCs alone with C792 does not affect the viability of either cell type. In agreement with our in vitro data, C792 inhibits pDC-induced MM cell growth in vivo in a murine xenograft model of human MM. Mechanistic studies show that C792 triggers maturation of pDCs, enhances interferon-α and interferon-λ secretion and activates TLR-9/MyD88 signaling axis. Finally, C792 enhances the anti-MM activity of bortezomib, lenalidomide, SAHA or melphalan. Collectively, our preclinical studies provide the basis for clinical trials of C792, either alone or in combination, to both improve immune function and overcome drug resistance in MM.

Evaluation of pectinolytic activities for oenological uses from psychrotrophic yeasts.

UNLABELLED: Of the twenty-three morphotypes of yeasts isolated from soil capable of utilizing pectin as sole carbon source at 6°C, two yeast isolates, one psychrotolerant (PT1) and one psychrophilic (SPY11), were selected according to their ability to secrete pectinolytic enzymes under some oenological conditions (temperature 6 and 12°C and pH 3.5) and ability or inability to grow above 20°C, respectively. As compared to their optimal activity, the three pectinolytic enzymes viz., pectin methyl esterase (PME), endopolygalacturonase (endo-PG) and exopolygalacturonase (exo-PG) isolated and assayed at pH 3.5 from PT1 were found to retain 39, 60 and 60% activity at 12°C and 40, 79 and 74% activity at 28°C, respectively. Likewise, the enzymes PME and endo-PG at pH 3.5 from SPY11 displayed 46 and 86% activity at 12°C and 50 and 60% activity at 28°C, respectively. All these enzymes showed 20-90% of residual activity at pH 3.5 and 6°C. The yeast isolates PT1 and SPY11 were identified as Rhodotorula mucilaginosa and Cystofilobasidium capitatum, respectively, on the basis of morphological, physiological and molecular characteristics. This study presents the first report on pectinolytic activities under major oenological conditions from psychrotolerant isolate R. mucilaginosa PT1 and psychrophilic isolate C. capitatum SPY11. SIGNIFICANCE AND IMPACT OF THE STUDY: The cold-active pectinolytic enzymes (PME, endo-PG and exo-PG) from the newly isolated and identified psychrophilic yeast Cystofilobasidium capitatum SPY11 and psychrotolerant yeast Rhodotorula mucilaginosa PT1that exhibited 50-80% of their optimum activity under some major oenological conditions pH (3.5) and temperatures (6 and 12°C) could be applied to wine production and juice clarification at low temperature. The psychrotrophic yeasts themselves could be applied to cold process for the production of enzymes thus saving cost of energy and protecting process from contamination.

Does it matter how we measure metatarsal length?

INTRODUCTION: Variations in the relative lengths of the metatarsal bones have been implicated as a cause of forefoot pain. Osteotomies to balance the metatarsal lengths have been described as therapy. Although measurement of metatarsal length is required for these osteotomies, there is no gold standard method to measure it radiologically. We compared the three main radiological methods to measure the second metatarsal length described in the literature. MATERIALS AND METHODS: Standing dorsoplantar radiographs of 81 feet were measured using the methods described by Coughlin, Maestro and Hardy and Clapham. Bland and Altman's method was used to compare the above techniques for reproducibility and agreement between each method. RESULTS: The three methods produced markedly different results. Coughlin's and Maestro's methods showed a relative protrusion of the second metatarsal-relatively longer than the first metatarsal by an average of 4 & 3 mm respectively. Hardy and Clapham's method, however, showed a relative retraction of second metatarsal by an average of 1 mm. Both intra-observer and inter-observer errors were smallest for the Coughlin method (the most reproducible) and largest for the Hardy/Clapham method. Significant lack of agreement was found when we compared the 3 techniques with bias for measuring the mean between methods ranging from 1mm to 5.5mm. CONCLUSIONS: Metatarsal lengths as measured by these three methods can be very variable. Thus their role in planning metatarsal osteotomies and establishing relationship of metatarsal protrusion with metatarsophalangeal joint instability and other similar conditions is questionable and require further studies.

Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.