Exosomes Represent an Immune Suppressive T Cell Checkpoint in Human Chronic Inflammatory Microenvironments.

Background: T cells present in chronic inflammatory tissues such as nasal polyps (from chronic rhinosinusitis patients) have been demonstrated to be hypo-responsive to activation via the TCR, similar to tumor-specific T cells in multiple different human tumor microenvironments. While immunosuppressive exosomes have been known to contribute to the failure of the tumor-associated T cells to respond optimally to activation stimuli, it is not known whether they play a similar role in chronic inflammatory microenvironments. In the current study, we investigate whether exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. Methods: Exosomes were isolated by ultracentrifugation and characterized by size and composition using nanoparticle tracking analysis, scanning electron microscopy, antibody arrays and flow exometry. Immunosuppressive ability of the exosomes was measured by quantifying its effect on activation of T cells, using nuclear translocation of NFκB as an activation endpoint. Results: Exosomes were isolated and characterized from two different types of chronic inflammatory tissues - nasal polyps from chronic rhinosinusitis patients and synovial fluid from rheumatoid arthritis patients. These exosomes arrest the activation of T cells stimulated via the TCR. This immune suppression, like that which is seen in tumor microenvironments, is dependent in part upon a lipid, ganglioside GD3, which is expressed on the exosomal surface. Conclusion: Immunosuppressive exosomes present in non-malignant chronic inflammatory tissues represent a new T cell checkpoint, and potentially represent a novel therapeutic target to enhance the response to current therapies and prevent disease recurrences.

Acridine form IX.

We report a new polymorph of acridine, C13H9N, denoted form IX, obtained as thin needles by slow evaporation of a toluene solution. The structure was solved and refined from powder X-ray data. The structures of five unsolvated forms were previously known, but this is only the second with one mol-ecule in the asymmetric unit. The melting point [differential scanning calorimetry (DSC) onset] and heat of fusion are 108.8 (3) °C and 19.2 (4) kJ mol-1, respectively.

A new polymorph of ammonium succinate - serendipity in action.

Succinic acid has been known since 1546 and was first chemically identified in the mid-19th century. In an attempt to prepare a molecular salt of succinic acid with (S)-(-)-α-methylbenzylamine, we have obtained the second polymorph of the monoammonium salt of succinic acid, NH4+·C4H5O4-. The crystal structure determination proves the structure of the ionic compound and the intimate role of the ammonium ion in the structure, which is compared to the earlier described polymorph.

Crystallographic education in the 21st century.

There are many methods that can be used to incorporate concepts of crystallography into the learning experiences of students, whether they are in elementary school, at university or part of the public at large. It is not always critical that those who teach crystallography have immediate access to diffraction equipment to be able to introduce the concepts of symmetry, packing or molecular structure in an age- and audience-appropriate manner. Crystallography can be used as a tool for teaching general chemistry concepts as well as general research techniques without ever having a student determine a crystal structure. Thus, methods for younger students to perform crystal growth experiments of simple inorganic salts, organic compounds and even metals are presented. For settings where crystallographic instrumentation is accessible (proximally or remotely), students can be involved in all steps of the process, from crystal growth, to data collection, through structure solution and refinement, to final publication. Several approaches based on the presentations in the MS92 Microsymposium at the IUCr 23rd Congress and General Assembly are reported. The topics cover methods for introducing crystallography to undergraduate students as part of a core chemistry curriculum; a successful short-course workshop intended to bootstrap researchers who rely on crystallography for their work; and efforts to bring crystallography to secondary school children and non-science majors. In addition to these workshops, demonstrations and long-format courses, open-format crystallographic databases and three-dimensional printed models as tools that can be used to excite target audiences and inspire them to pursue a deeper understanding of crystallography are described.

Facts and fictions about polymorphism.

We present new facts about polymorphism based on (i) crystallographic data from the Cambridge Structural Database (CSD, a database built over 50 years of community effort), (ii) 229 solid form screens conducted at Hoffmann-La Roche and Eli Lilly and Company over the course of 8+ and 15+ years respectively and (iii) a dataset of 446 polymorphic crystals with energies and properties computed with modern DFT-d methods. We found that molecular flexibility or size has no correlation with the ability of a compound to be polymorphic. Chiral molecules, however, were found to be less prone to polymorphism than their achiral counterparts and compounds able to hydrogen bond exhibit only a slightly higher propensity to polymorphism than those which do not. Whilst the energy difference between polymorphs is usually less than 1 kcal mol(-1), conformational polymorphs are capable of differing by larger values (up to 2.5 kcal mol(-1) in our dataset). As overall statistics, we found that one in three compounds in the CSD are polymorphic whilst at least one in two compounds from the Roche and Lilly set display polymorphism with a higher estimate of up to three in four when compounds are screened intensively. Whilst the statistics provide some guidance of expectations, each compound constitutes a new challenge and prediction and realization of targeted polymorphism still remains a holy grail of materials sciences.

Disappearing polymorphs revisited.

Nearly twenty years ago, Dunitz and Bernstein described a selection of intriguing cases of polymorphs that disappear. The inability to obtain a crystal form that has previously been prepared is indeed a frustrating and potentially serious problem for solid-state scientists. This Review discusses recent occurrences and examples of disappearing polymorphs (as well as the emergence of elusive crystal forms) to demonstrate the enduring relevance of this troublesome, but always captivating, phenomenon in solid-state research. A number of these instances have been central issues in patent litigations. This Review, therefore, also highlights the complex relationship between crystal chemistry and the law.

"Predicting" Polymorphs of Pharmaceuticals Using Hydrogen Bond Propensities: Probenecid and Its Two Single-Crystal-to-Single-Crystal Phase Transitions.

The recently developed hydrogen-bonding propensity tool in the Cambridge Structural Database software package (Mercury) was tested to predict polymorphs. The compounds for the study were chosen from a list of approximately 300 pharmaceutically important compounds, for which multiple crystal forms had not been previously reported. The hydrogen-bonding propensity analysis was carried out on approximately 60 randomly selected compounds from this list. Several compounds with a high probability for exhibiting polymorphism in the analysis were chosen for a limited experimental crystal form screening. One of the compounds, probenecid, did not yield polymorphs by traditional solution crystallization screening, but differential scanning calorimetry revealed three polymorphs. All of them exhibit the same hydrogen bonding and transform via two reversible single-crystal-to single-crystal transformations, which have been characterized in detail through three single-crystal structure determinations at appropriate temperatures.

Kitaigorodsky revisited: polymorphism and mixed crystals of acridine/phenazine.

Non-stoichiometric molecular mixed crystals have potential as functional materials, the properties of which can be tailored by rationally changing their composition. The guidelines for their preparation were summarized over thirty years ago by Alexander Kitaigorodsky. Here those principles are revised in light of new studies on the acridine/phenazine system, and solvent-assisted grinding is presented as a convenient synthetic procedure to afford a more homogeneous product than traditional solvent-evaporation methods. Finally, the proposed prerequisite of crystal isostructurality/isomorphicity for the pure compounds, which seems to be violated in the present case, is discussed.

On the propulsion mechanism of "jumping" crystals.

A crystalline metal-organic complex undergoes a polymorphic transformation upon cooling below 215 K. Depending on the cooling rate, crystals exhibit different behaviours that include breaking and "jumping". Variable temperature X-ray diffraction (XRD) and optical microscopy have been employed to analyze the anisotropic changes of the crystallographic axes and propose a mechanism by which the crystals acquire the momentum for the jump.

The elusive crystal structure of the neuraminidase inhibitor Tamiflu (oseltamivir phosphate): molecular details of action.

The first structure determination of a neuraminidase inhibitor, oseltamivir phosphate, the active component of the anti-influenza agent Tamiflu, was achieved by single crystal X-ray diffraction with synchrotron radiation. The structure reproduces to a great extent the binding of the inhibitor in the neuraminidase active site and provides more accurate structural parameters as well as detailed insight into the binding preferences of the inhibitor.

Rietveld refinement of the cocrystal 2,4-dihydroxybenzoic acid-N'-(propan-2-ylidene)nicotinohydrazide (1/1).

A further example of using a covalent-bond-forming reaction to alter supramolecular assembly by modification of hydrogen-bonding possibilities is presented. This concept was introduced by Lemmerer, Bernstein & Kahlenberg [CrystEngComm (2011), 13, 55-59]. The title structure, C(9)H(11)N(3)O·C(7)H(6)O(4), which consists of a reacted niazid molecule, viz. N'-(propan-2-ylidene)nicotinohydrazide, and 2,4-dihydroxybenzoic acid, was solved from powder diffraction data using simulated annealing. The results further demonstrate the relevance and utility of powder diffraction as an analytical tool in the study of cocrystals and their hydrogen-bond interactions.

Humanized mouse model used to monitor MUC gene expression in nasal polyps and to preclinically evaluate the efficacy of montelukast in reducing mucus production.

OBJECTIVES: To determine whether MUC gene expression could be down-regulated in nasal polyps by the leukotriene receptor antagonist montelukast, we developed a system in which nondisrupted human nasal polyps could be successfully implanted into severely immunocompromised mice, and in which the histopathology of the original nasal polyp tissue could be preserved for long periods. In addition, the histopathologic changes in the human nasal polyps were carefully examined to determine the origin of the submucosal glands (SMGs) that develop in true nasal polyps found in the anterior third of the nose. METHODS: Small, nondisrupted pieces of human nasal polyp tissues were subcutaneously implanted into NOD-scid IL-2rgamma(null) mice. Xenograft-bearing mice were treated with either montelukast or saline solution. Xenografts at 8 to 12 weeks after implantation were examined histologically, and expression of MUC genes 4, 5AC, and 7 was studied in the polyps before implantation and in the 8-week xenograft. Alzet pumps were inserted into the mice, and montelukast (Singulair) was continuously delivered to determine its effect on goblet cell hyperplasia, mucus production, and the enlargement of nasal polyps over an 8-week period. RESULTS: The xenografts were maintained in a viable and functional state for up to 3 months and retained a histopathology similar to that of the original tissue, but with a noticeable increase in goblet cell hyperplasia and marked mucus accumulation in the SMGs. MUC4 and MUC5AC were significantly increased in the xenograft 8 weeks after implantation, but MUC7 was significantly decreased compared to the preimplantation polyps. Inasmuch as MUC7 is found exclusively in serous glands, the findings suggest that serous glands are not found in polyps in the anterior third of the nose. The histopathologic findings confirm the original findings of Tos et al suggesting that the SMGs are derived from pinching-off of the epithelium of the enlarging polyp following inflammatory changes. These SMGs have the same epithelium as surface epithelium and consist of multiple goblet cells that secrete periodic acid Schiff stain-positive mucin into the interior of the SMGs. A progressive increase in the volume of the xenografts was observed, with little or no evidence of mouse cell infiltration into the human leukocyte antigen-positive human tissue. An average twofold increase in polyp volume was found 2 months after engraftment. Montelukast did not decrease the growth of the xenograft in the 8-week NOD-scid mice, nor did it affect MUC gene expression. CONCLUSIONS: The use of innate and adaptive immunodeficient NOD-scid mice homozygous for targeted mutations in the IL-2 gamma-chain locus NOD-scid IL-2r gamma(null) for establishing engraftment of nondisrupted pieces of human nasal polyp tissues represents a significant advancement in studying chronic inflammation over a long period of time. In the present study, we utilized this humanized mouse model to confirm our prediction that MUC genes 4 and 5AC are highly expressed and significantly increased over those of preimplanted polyps. The overexpression of these 2 MUC genes correlates with both the goblet cell hyperplasia and the excessive mucus production that are found in nasal polyp xenografts. MUC7, which is primarily associated with the submucosa, as opposed to MUC4 and MUC5AC, which are primarily expressed in the epithelium, was significantly decreased in the nasal polyp xenografts. Montelukast had no significant effect on MUC gene expression in the xenografts. In addition to the MUC gene expression patterns, the histology of the xenografts supports the concept that mucinous glands that are characteristic of true nasal polyps are significantly different from those in the mucosa found in the lateral wall of the nose in patients with chronic sinusitis without nasal polyps. The mucinous glands seen in nasal polyps (which appear to be derived from an invagination of hyperplastic epithelial mucosa containing large numbers of goblet cells) are histologically distinct from the seromucinous glands found in the submucosa of hyperplastic middle turbinates. The data presented here establish a humanized mouse model as a viable approach to study nasal polyp growth, to assess the therapeutic efficacy of various drugs in this chronic inflammatory disease, and to contribute to our understanding of the pathogenesis of this disease.

N'-Cyclo-pentyl-idene-pyridine-4-carbo-hydrazide.

The title compound, C(11)H(13)N(3)O, is a derivative of the anti-tuberculosis drug isoniazid [systematic name: pyridine-4-carbohydrazide]. The crystal structure consists of repeating hydrogen-bonded chains parallel to the b axis. Adjacent mol-ecules in the chains are linked by bifurcated N-H⋯(O,N) hydrogen bonds, which form an R(1) (2)(5) ring motif.

N'-Cyclo-dodecyl-idene-pyridine-4-carbohydrazide.

The title compound, C(18)H(27)N(3)O, is a derivative of the anti-tuberculosis drug isoniazid (systematic name: pyridine-4-carbohydrazidei). The crystal structure consists of repeating C(4) chains along the b axis, formed by N-H⋯O hydrogen bonds with adjacent amide functional groups that are related by a b-glide plane. The cyclo-dodecyl ring has the same approximately 'square' conformation, as seen in the parent hydro-carbon cyclo-dodecane.

Memory T cells in the chronic inflammatory microenvironment of nasal polyposis are hyporesponsive to signaling through the T cell receptor.

A majority of T cells from chronic inflammatory tissues derived from patients with nasal polyposis were found to express an effector memory phenotype. We report here that these memory T cells failed to activate NF-κB in response to TCR stimulation but responded normally when the proximal TCR signaling molecules were bypassed with PMA and ionomycin. The dysfunction of these cells was associated with a decrease in the phosphorylation of several TCR proximal signaling molecules including ZAP70, Lck and SLP-76. In addition to the disruption in the TCR signaling pathway, the nasal polyp-associated T cells were shown to have a defect in their ability to translocate LAMP-1 to the cell surface. The results presented here establish that the phenotype and anergy of the T cells in the nasal polyp are similar to those which is seen in memory T cells derived from human tumors and other sites of chronic inflammation.

Form II of adipic acid-nicotinohydrazide (1/2).

The crystal structure of the title co-crystal, 2C(6)H(7)N(3)O·C(6)H(10)O(4), is a second polymorph, designated form II, of the co-crystal formed between the two mol-ecules [Lemmerer et al. (2011 ▶). CrystEngComm, 13, 55-59]. The asymmetric unit comprises one mol-ecule of nicotinic acid hydrazide, and one half-mol-ecule of adipic acid (the entire mol-ecule is completed by the application of a centre of inversion). In the crystal, mol-ecules assemble into a three-dimensional network of hydrogen bonds, formed by three N-H⋯O hydrogen bonds and one O-H⋯N hydrogen bond. The O-H⋯N hydrogen bond formed between the carboxyl group and the pyridine ring is supported by a C-H⋯O hydrogen bond.

Civamide cream 0.075% in patients with osteoarthritis of the knee: a 12-week randomized controlled clinical trial with a longterm extension.

OBJECTIVE: To evaluate the safety and efficacy of civamide cream 0.075% for the treatment of osteoarthritis (OA) of the knee. METHODS: We conducted a 12-week, multicenter, randomized, double-blind study with a 52-week open-label extension. Patients with OA of the knee received either civamide cream 0.075% or a lower dose of civamide cream, 0.01%, as the control. The 3 co-primary endpoints in the double-blind study were the time-weighted average (TWA) of change from baseline to Day 84 in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscale, the WOMAC physical function subscale, and the Subject Global Evaluation (SGE). In the 52-week open-label extension study, the Osteoarthritis Pain Score and SGE were assessed. RESULTS: A total of 695 patients were randomized to receive civamide cream 0.075% (n = 351) or civamide cream 0.01% (control; n = 344) in the double-blind study. Significance in favor of civamide cream 0.075% was achieved for the TWA for all 3 co-primary efficacy variables: WOMAC pain (p = 0.009), WOMAC physical function (p < 0.001), and SGE (p = 0.008); and at Day 84 for these 3 variables (p = 0.013, p < 0.001, and p = 0.049, respectively). These analyses accounted for significant baseline-by-treatment interactions. In the 52-week open-label extension, efficacy was maintained. Civamide cream 0.075% was well tolerated throughout the studies. CONCLUSION: These studies demonstrate the efficacy of civamide cream for up to 1 year of continuous use. Civamide cream, with its lack of systemic absorption, does not have the potential for serious systemic toxicity, in contrast to several other OA treatments.

Supramolecular polymorphism of the 1:1 molecular salt (adamantane-1-carboxylate-3,5,7-tricarboxylic acid)·(hexamethylenetetraminium). A "failed" crystal engineering attempt.

The tetrahedral arrangement of hydrogen bonding donor and acceptor groups is used to rationalise the design of a diamondoid network; however, a single proton transfer renders the four sites inequivalent, and results in two polymorphs of the title molecular salt utilizing similar intermolecular synthons.

A tale of two polymorphic pharmaceuticals: pyrithyldione and propyphenazone and their 1937 co-crystal patent.

A co-crystal of two polymorphic active pharmaceutical ingredients (APIs), first reported and patented in 1937, has been prepared and thoroughly characterised, including crystal structure analysis. The existence of four crystal forms of one of the APIs, the sedative and hypnotic active pharmaceutical ingredient 3,3-diethyl-2,4(1H,3H)-pyridinedione, pyrithyldione (PYR), and of three crystal forms of the co-crystal-forming second API, the non-steroidal anti-inflammatory drug 1,2-dihydro-1,5-dimethyl-4-(1-methylethyl)-2-phenyl-3H-pyrazol-3-one, propyphenazone (PROP), has been reported previously, but they have only been partly characterised. For both compounds, none of the metastable forms exist at room temperature. DSC, hot-stage microscopy, X-ray diffraction and powder synchrotron X-ray diffraction were employed to characterise the polymorphic forms and to determine the crystal structures of forms I-III of PYR and forms I and II of PROP.