Spatial genomic heterogeneity in multiple myeloma revealed by multi-region sequencing.

In multiple myeloma malignant plasma cells expand within the bone marrow. Since this site is well-perfused, a rapid dissemination of "fitter" clones may be anticipated. However, an imbalanced distribution of multiple myeloma is frequently observed in medical imaging. Here, we perform multi-region sequencing, including iliac crest and radiology-guided focal lesion specimens from 51 patients to gain insight into the spatial clonal architecture. We demonstrate spatial genomic heterogeneity in more than 75% of patients, including inactivation of CDKN2C and TP53, and mutations affecting mitogen-activated protein kinase genes. We show that the extent of spatial heterogeneity is positively associated with the size of biopsied focal lesions consistent with regional outgrowth of advanced clones. The results support a model for multiple myeloma progression with clonal sweeps in the early phase and regional evolution in advanced disease. We suggest that multi-region investigations are critical to understanding intra-patient heterogeneity and the evolutionary processes in multiple myeloma.In multiple myeloma, malignant cells expand within bone marrow. Here, the authors use multi-region sequencing in patient samples to analyse spatial clonal architecture and heterogeneity, providing novel insight into multiple myeloma progression and evolution.

A novel flexible cuff-like microelectrode for dual purpose, acute and chronic electrical interfacing with the mouse cervical vagus nerve.

OBJECTIVE: Neural reflexes regulate immune responses and homeostasis. Advances in bioelectronic medicine indicate that electrical stimulation of the vagus nerve can be used to treat inflammatory disease, yet the understanding of neural signals that regulate inflammation is incomplete. Current interfaces with the vagus nerve do not permit effective chronic stimulation or recording in mouse models, which is vital to studying the molecular and neurophysiological mechanisms that control inflammation homeostasis in health and disease. We developed an implantable, dual purpose, multi-channel, flexible 'microelectrode' array, for recording and stimulation of the mouse vagus nerve. APPROACH: The array was microfabricated on an 8 µm layer of highly biocompatible parylene configured with 16 sites. The microelectrode was evaluated by studying the recording and stimulation performance. Mice were chronically implanted with devices for up to 12 weeks. MAIN RESULTS: Using the microelectrode in vivo, high fidelity signals were recorded during physiological challenges (e.g potassium chloride and interleukin-1ß), and electrical stimulation of the vagus nerve produced the expected significant reduction of blood levels of tumor necrosis factor (TNF) in endotoxemia. Inflammatory cell infiltration at the microelectrode 12 weeks of implantation was limited according to radial distribution analysis of inflammatory cells. SIGNIFICANCE: This novel device provides an important step towards a viable chronic interface for cervical vagus nerve stimulation and recording in mice.

The haptoglobin beta subunit sequesters HMGB1 toxicity in sterile and infectious inflammation.

BACKGROUND: Extra-corpuscular haemoglobin is an endogenous factor enhancing inflammatory tissue damage, a process counteracted by the haemoglobin-binding plasma protein haptoglobin composed of alpha and beta subunits connected by disulfide bridges. Recent studies established that haptoglobin also binds and sequesters another pro-inflammatory mediator, HMGB1, via triggering CD163 receptor-mediated anti-inflammatory responses involving heme oxygenase-1 expression and IL-10 release. The molecular mechanism underlying haptoglobin-HMGB1 interaction remains poorly elucidated. METHODS: Haptoglobin ß subunits were tested for HMGB1-binding properties, as well as efficacy in animal models of sterile liver injury (induced by intraperitoneal acetaminophen administration) or infectious peritonitis (induced by cecal ligation and puncture, CLP, surgery) using wild-type (C57BL/6) or haptoglobin gene-deficient mice. RESULTS: Structural-functional analysis demonstrated that the haptoglobin ß subunit recapitulates the HMGB1-binding properties of full-length haptoglobin. Similar to HMGB1-haptoglobin complexes, the HMGB1-haptoglobin ß complexes also elicited anti-inflammatory effects via CD163-mediated IL-10 release and heme oxygenase-1 expression. Treatment with haptoglobin ß protein conferred significant protection in mouse models of polymicrobial sepsis as well as acetaminophen-induced liver injury, two HMGB1-dependent inflammatory conditions. CONCLUSIONS: Haptoglobin ß protein offers a novel therapeutic approach to fight against various inflammatory diseases caused by excessive HMGB1 release.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Ni(II) and Zn(II) Complexes Containing Alkynyl Functionalized Salicylaldimine Ligand and Heterocyclic Coligand: Synthesis, Characterization and Luminescence Properties.

Some nickel(II) and zinc(II) complexes of the type [Ni(L)(phen/bipy)]X (1a-6a) and [Zn(L) (phen/bipy)]X (1b-6b) (where L = 2-{(E)-[(4-trimethylsilylethynylphenyl)imino]methyl}-4-(4-nitro phenylethynyl)phenol; phen = 1, 10-phenanthroline, bipy = 2, 2´-bipyridine; X = ClO4-, BF4-, PF6-) have been prepared and characterized on the basis of elemental analyses, FTIR, 1H NMR and mass spectral studies. The molecular structure of L was determined by single crystal X-ray diffraction studies. The electrochemical behaviour of the Ni(II) complexes indicate that the phen complexes appears at more positive potential as compared to those for bipy complexes, as a consequence of its strong π-acidic character. TGA was carried out to study the thermal behavior of the complexes. Room temperature luminescence is observed for all complexes corresponds to π â†’ π* ILCT transition. The size of the counter anion and heterocyclic coligands phen and bipy shows marked effect on emission properties of the complexes.

Copper(I) Complexes of N-(2-{[(2E)-2-(4-Nitrobenzylidenyl)Hydrazinyl]Carbonyl}Phenyl)Benzamide and Triphenylphosphine: Synthesis, Characterization and Luminescence Properties.

Copper(I) complexes of the formula [Cu(L)(PPh3)2]X (1-4) (X = Cl(1), ClO4(2), BF4(3) and PF6(4)) [where L = N-(2-{[(2E)-2-(4-nitrobenzylidenyl)hydrazinyl]carbonyl}phenyl)benzamide; PPh3 = triphenylphosphine] have been prepared by the condensation of N-[2-(hydrazinocarbonyl)phenyl]benzamide with 4-nitrobenzaldehyde followed by the reaction with CuCl, [Cu(MeCN)4]ClO4, [Cu(MeCN)4]BF4 and [Cu(MeCN)4]PF6 in presence of triphenylphosphine as a coligand. Complexes 1-4 were then characterized by elemental analyses, FTIR, UV-visible and 1H NMR spectroscopy. Mononuclear copper(I) complexes 1-4 were formed with L in its keto form by involvement of azomethine nitrogen and the carbonyl oxygen along with two PPh3 groups. A single crystal X-ray diffraction study of the representative complex [(Cu(L)(PPh3)2]CIO4 (2) reveals a distorted tetrahedral geometry around Cu(I). Crystal data of (2): space group = C2/c, a = 42.8596 (9) Å, b = 14.6207 (3) Å, c = 36.4643 (7) Å, V = 20,653.7 (7) Å3, Z = 16. Complexes 1-4 exhibit quasireversible redox behaviour corresponding to a Cu(I)/Cu(II) couple. All complexes show blue-green emission as a result of fluorescence from an intra-ligand charge transition (ILCT), ligand to ligand charge transfer transition (LLCT) or mixture of both. Significant increase in size of the counter anion shows marked effect on quantum efficiency and lifetime of the complexes in solution.

Copper(II) complexes of N-(2-{[(2E)-2-(2-Hydroxy-(5-substituted)-benzylidene)-hydrazino]carbonyl}phenyl)benzamide ligands and heterocyclic coligands.

Some copper(II) complexes of the type [Cu(L1-3)(phen]·CH2Cl2 (1a-3a) and [Cu(L1-3) (bipy)]·CH2Cl2 (1b-3b) (where L1=N-(2-{[(2E)-2-(2-Hydroxy-benzylidene)-hydrazino]carbonyl}phenyl)benzamide, L2=N-(2-{[(2E)-2-(2-Hydroxy-(5-bromo)-benzylidene)-hydrazino]carbonyl}phenyl)benzamide, L3=N-(2-{[(2E)-2-(2-Hydroxy-(5-methoxy)-benzylidene)-hydrazino]carbonyl}phenyl)benzamide; phen=1,10-phenanthroline, bipy=2,2'-bipyridine) have been prepared and characterized on the basis of elemental analyses, IR, UV-Vis and EPR spectral studies. IR spectra indicate that the ligand L1-3 exists in the keto form in the solid state, while at the time of complexation, it tautomerises into enol form. The single crystal X-ray diffraction study of the representative complex [Cu(L1) (phen)]·CH2Cl2 (1a) reveals the distorted square pyramidal geometry around copper(II). Crystal data of (1a): space group=P21/n, a=11.5691(16) Å, b=11.0885(15) Å, c=24.890(4) Å, V=3166.2(8) Å(3), Z=4. The electrochemical behavior of all the complexes indicate that the phen complexes appears at more positive potential as compared to those for bipy complexes, as a consequence of its stronger π acidic character. All the complexes exhibit blue-green emission as a result of the fluorescence from the intra-ligand (π→π(*)) emission excited state.

HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly-6C(high) inflammatory monocytes in murine sepsis survivors.

BACKGROUND: More than 500,000 hospitalized patients survive severe sepsis annually in the USA. Recent epidemiological evidence, however, demonstrated that these survivors have significant morbidity and mortality, with 3-year fatality rates higher than 70%. To investigate the mechanisms underlying persistent functional impairment in sepsis survivors, here we developed a model to study severe sepsis survivors following cecal ligation and puncture (CLP). METHODS: Sepsis was induced in mice by CLP and survivors were followed for twelve weeks. Spleen and blood were collected and analyzed at different time points post-sepsis. RESULTS: We observed that sepsis survivors developed significant splenomegaly. Analysis of the splenic cellular compartments revealed a major expansion of the inflammatory CD11b+ Ly-6CHigh pool. Serum high-mobility group box 1 (HMGB1) levels in the sepsis surviving mice were significantly elevated for 4-6 weeks after post-sepsis, and administration of an anti-HMGB1 monoclonal antibody significantly attenuated splenomegaly as well as splenocyte priming. Administration of recombinant HMGB1 to naive mice induced similar splenomegaly, leukocytosis and splenocyte priming as observed in sepsis survivors. Interestingly analysis of circulating HMGB1 from sepsis survivors by mass spectroscopy demonstrated a stepwise increase of reduced form of HMGB1 (with known chemo-attractant properties) during the first 3 weeks, followed by disulphide form (with known inflammatory properties) 4-8 weeks after CLP. DISCUSSION: Our results indicate that prolonged elevation of HMGB1 is a necessary and sufficient mediator of splenomegaly and splenocyte expansion, as well as splenocyte inflammatory priming in murine severe sepsis survivors.

Synthesis, spectral characterization, thermal and photoluminescence properties of Zn(II) and Cd(II)-azido/thiocyanato complexes with thiazolylazo dye and 1,2-bis(diphenylphoshino)ethane.

A series of complexes of the type [M(L)(dppe)X2]; where M=Zn(II) or Cd(II); L=4-(2'-thiazolylazo)chlorobenzene (L1), 4-(2'-thiazolylazo)bromobenzene (L2) and 4-(2'-thiazolylazo) iodobenzene (L3); dppe=1,2-bis(diphenylphosphino)ethane; X=N3- or NCS- have been prepared and characterized on the basis of their microanalysis, molar conductance, thermal, IR, UV-vis and 1H NMR spectral studies. IR spectra show that the ligand L is coordinated to the metal atom in bidentate manner via azo nitrogen and thiazole nitrogen. An octahedral structure is proposed for all the complexes. The thermal behavior of the complexes revealed that the thiocyanato complexes are thermally more stable than the azido complexes. All the complexes exhibit blue-green emission with high quantum yield as the result of the fluorescence from the intraligand emission excited state.

Synthesis, structural characterization, thermal and electrochemical studies of mixed ligand Cu(II) complexes containing 2-phenyl-3-(benzylamino)-1,2-dihydroquinazoline-4-(3H)-one and bidentate N-donor ligands.

Some mixed ligand Cu(II) complexes of the type [Cu(L)(en)X(2)] (1a-3a), [Cu(L)(en)](ClO(4))(2) (4a), [Cu(L)(phen)X(2)] (1b-3b) and [Cu(L)(phen)](ClO(4))(2) (4b) [where L = 2-phenyl-3-(benzylamino)-1,2-dihydroquinazoline-4-(3H)-one; en = ethylenediamine; phen = 1,10-phenanthroline; X = Cl(-), N(3)(-) and NCS(-)] have been prepared. The complexes were characterized on the basis of elemental analysis, molar conductance, magnetic moment, IR, UV-vis, mass, ESR and thermal studies. On the basis of electronic spectral data and magnetic susceptibility measurement octahedral geometry has been proposed for 1a-3a and 1b-3b and square-planer geometry for 4a and 4b. The ESR spectral data of complexes provided information about their structure on the basis of Hamiltonian parameters and degree of covalency. The electrochemical behaviour of mixed ligand Cu(II) complexes was studied which showed that complexes of phen appear at more positive potential as compared to those for corresponding en complexes.

Characterization and electrochemical studies of Mn(II), Co(II), Ni(II) and Cu(II) complexes with 2-mercapto-3-substituted-quinazolin-4-one and 1,10-phenanthroline or ethylenediamine as ligands.

Some mixed ligand complexes of the type [M(L(1) or L(2))(phen or en)(H(2)O)Cl], where M=Mn(II), Co(II), Ni(II) and Cu(II); HL(1)=2-mercapto-quinazolin-4-one; HL(2)=2-mercapto-3-phenyl-quinazolin-4-one; phen=1,10-phenanthroline; en=ethylenediamine have been prepared. All complexes were characterized on the basis of elemental analysis, molar conductance, magnetic susceptibility measurements, IR, UV-vis, ESR and powder X-ray diffraction studies. IR spectra of these complexes reveal that the complex formation occurred through both nitrogen and sulphur atoms. On the basis of electronic spectral data and magnetic susceptibility measurement octahedral geometry has been proposed for the complexes. The ESR spectral data of the Cu(II) complexes showed that the metal-ligand bonds have considerable covalent character. X-ray diffraction studies of Cu(II) complexes are used to elucidate the crystal structure. The electrochemical behaviour of mixed ligand Ni(II) complexes was studied which showed that complexes of phen appear at more positive potential as compared to those for corresponding en complexes.

Rhinosporidiosis--a clinicopathological study of 34 cases.

Rhinosporidiosis was the commonest (68%) fungal lesion encountered during the period of 11 1/2 years from January 1987 to July 1998. Men in 2nd, 3rd, 4th decade were commonly affected. Nose and nasopharynx were the commonest (85%) sites involved followed by ocular tissue (9%). One patient had involvement of bone (tibia). Generally a lymphoplasmacytic response was observed in all cases. Polymorphonuclear leukocytic response mostly observed at the site of rupture of sporangia. Epithelioid cell granulomatous and giant cell response observed in 47% of cases. Transepithelial migration of sporangia observed in 76% of cases. Rhinosporidium seeberi could be easily identified in haematoxylin and eosin stained sections. The walls of young trophic forms are delineated well with the PAS stain and verhoeff van Gieson stain.

Clinical profile of non-O1 strain-O139 of Vibrio cholerae in the region of Ambajogai, Maharashtra.

OBJECTIVES: To study clinical profile of the newly emerged novel strain non-O1, O139 of Vibrio cholerae, in the region of Ambajogai, District Beed of Maharashtra. METHODS: Out of 208 patients of acute gastroenteritis, 41 revealed to be positive for Vibrio cholerae by recommended method of stool examination. All the strains were sent to National Institute of Cholera and Infectious Diseases, Calcutta for confirmation. RESULTS: Out of 41 cases, 12 were of Vibrio cholerae O1, 29 Non-O1, of which nine found to be O139 strain. All patients were from 2-80 years of age with low-socioeconomic status and maximum incidence was in August (64.70%), presented with severe rice watery loose motions. Vomiting was observed in 26 (63.41%), more so in patients of O139 infection (88.88%) than four (33.33%) of O1 infection. Sweating was observed in three patients (33.33%) of O139 infection, cramps in gastrocnemis muscles in three patients (33.33%) of O139 infection and two (16.66%) of O1 infection. Signs of dehydration were mild to moderate in four patients (33.33%) of O1 infection; severe dehydration in six (66.66%), moderate in two (22.22%) and mild in one patient (11.11%) of O139 infection. While dehydration was severe in four (20%), moderate in one (5%) and mild in three patients (15%) of Non-O1 infection (excluding O139 cases). Clinical features were more severe in patients of serotype O139 than the patients of O1 and Non-O1 (excluding O139 cases). However all patients responded to intravenous fluids, oral rehydration and antibiotics (tetracycline) within 24-48 hours without any mortality. CONCLUSIONS: This study reflects the first emergence of Non-O1, strain O139 during the year 1997 with severe and critical clinical features in Ambajogai region causing high morbidity in the form of severe dehydration and peripheral circulatory collapse which requires early and correct diagnosis and prompt treatment.

Immunophenotypes and cytotoxic functions of lymphocytes in patients with hepatocellular carcinoma.

AIMS AND BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia. Immunological mechanisms are thought to play an important role in the control of tumor progression. The immune responses in HCC patients are poorly understood. In the present study, the proliferation and cytotoxic functions of lymphocytes from tumor tissues and peripheral blood of HCC patients were analysed. Simultaneously, the microcultures were phenotyped in order to determine the involvement of different lymphocyte subsets in mediating the cytotoxic function. METHODS: The frequencies of proliferating and cytotoxic lymphocytes from three tumor tissues and peripheral blood from ten HCC patients and nine healthy individuals were assessed by limiting dilution microculture analysis. These microcultures were phenotyped by single and dual color flow cytometry using monoclonal antibodies specific for CD4, CD8, CD56 and HLA-DR markers. RESULTS: The precursor frequencies of both proliferating and cytotoxic lymphocytes were found to be comparable in the peripheral blood of HCC patients and healthy individuals. Compared to peripheral blood, a marked reduction in the precursor frequencies of proliferating and cytotoxic lymphocytes was observed in the tumor tissues of HCC patients. In the tumor tissues, a significantly higher frequency of cytotoxic T cells compared to natural killer cells was observed. Dual color flow cytometric analysis revealed increased percentages of CD8+ HLA-DR+ lymphocytes compared to CD4+ HLA-DR+ cells in the tumor tissues. CONCLUSIONS: Our results suggest that depressed immune responses at the tumor site might be responsible for the escape of tumor cells from the immune surveillance of the host.

Frequency analysis of proliferating and cytotoxic T cells in livers and peripheral blood of patients with chronic hepatitis B.

Frequencies of proliferating and cytotoxic lymphocytes from liver biopsy samples and peripheral blood of chronic hepatitis B (CHB) patients and control subjects were monitored by limiting dilution analysis. Precursor frequencies of proliferating T lymphocytes were not significantly different in the liver and peripheral blood compartments of patients and controls. Moreover, similar frequencies of natural killer cells and cytotoxic T lymphocytes were observed in the peripheral blood of patients and controls. A higher frequency of cytotoxic T cells (1 of 22) compared to NK cells (1 of 306) was observed in liver tissues of CHB patients. Dual colour flow cytometric analysis revealed the presence of both CD4+ HLA-DR+ and CD8+ HLA-DR+ T cells in the liver tissues. These results suggest that in livers of CHB patients not only activated CD8+ T cells but also activated CD4+ T cells may play a significant role in the pathogenesis of chronic hepatitis B.