Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

The immunodeficiency of chronic lymphocytic leukaemia.

INTRODUCTION: Patients with chronic lymphocytic leukaemia (CLL) have progressive immunodeficiency and infection is the commonest cause of death. This review seeks to identify the extent of the abnormality, its cause, clinical significance and any possible remedy. SOURCES OF DATA: TJH has studied CLL for the past 40 years and has scanned or read every paper he could find published on the topic since 1970 and most of those of historical importance published before that date. He has read around the subject, covering relevant articles on immunology, cell biology, oncology and genetics. Furthermore, he has attended most major meetings dealing with CLL in this time and has written many reviews to update the state of knowledge about the topic. He receives weekly updates of papers published on CLL from PubMed and Science Direct with the keywords 'chronic lymphocytic leukaemia'. AREAS OF AGREEMENT: The immunodeficiency chiefly manifests as hypogammaglobulinaemia but involves all elements of the immune system. It is caused by the interpolation of tumour cells among immunological cells and mediated by bi-directional cell contact and secretion of cytokines, which both sustain and invigorate the tumour and suppress immunity. CLL treatment generally makes the immunodeficiency worse. Intravenous immunoglobulin is clinically effective but not cost-effective, while prophylactic antibiotics are useful in appropriate circumstances. Vaccination against infectious disease is usually ineffective. AREAS OF CONTROVERSY: Exactly how the presence of tumour cells in the immune organs renders the patient immunodeficient is controversial as is the clinical significance of minor degrees of immunodeficiency in early or indolent cases. The immunosuppressive effect of most forms of treatment is agreed, but how much this should figure in the choice of treatment is a matter of dispute. GROWING POINTS: The study of tumour-stromal interactions is an area of intense research. AREAS TIMELY FOR DEVELOPING RESEARCH: There has been little done to develop better vaccination strategies in patients with CLL, and although effective antimicrobials have been developed to protect against opportunistic infections, many are both expensive and inconvenient. More work is necessary to define precisely which patients should be offered them and when.

Provision of long-term monitoring and late effects services following adult allogeneic haematopoietic stem cell transplant: a survey of UK NHS-based programmes.

Despite international guidelines, optimal delivery models of late effects (LE) services for HSCT patients are unclear from the clinical, organizational and economic viewpoints. To scope current LE service delivery models within the UK NHS (National Health Service), in 2014, we surveyed the 27 adult allogeneic HSCT centres using a 30-question online tool, achieving a 100% response rate. Most LE services were led and delivered by senior physicians (>80% centres). Follow-up was usually provided in a dedicated allograft or LE clinic for the first year (>90% centres), but thereafter attrition meant that only ~50% of patients were followed after 5 years. Most centres (69%) had a standard operating procedure for long-term monitoring but access to a LE Multi-Disciplinary Team was rare (19% centres). Access to medical specialities necessary for LE management was good, but specialist interest in long-term HSCT complications was uncommon. Some screening (endocrinopathy, cardiovascular) was near universal, but other areas were more limited (mammography, cervical smears). Funding of extra staff and investigations were the most commonly perceived barriers to implementation of LE services. This survey shows variation in the long-term follow-up of allogeneic HSCT survivors within the UK NHS and further work is warranted to optimize effective, sustainable and affordable models of LE service delivery among this group.

Routine vaccination practice after adult and paediatric allogeneic haematopoietic stem cell transplant: a survey of UK NHS programmes.

This corrects the article DOI: 10.1038/bmt.2016.362.

Immunogenetic analysis reveals that epitope shifting occurs during B-cell affinity maturation in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a liver disease characterized by serum autoantibodies against the pyruvate dehydrogenase complex (PDC) located in the inner mitochondrial membrane. The predominant target in PDC has previously been localized to the inner lipoyl domain (ILD) of the E2 subunit. The etiology of PBC is unknown, although molecular mimicry with bacterial PDC has been proposed. Here, we have investigated the etiology of PBC and nature of the autoimmune response by analyzing the structure of a human monoclonal antibody with ILD specificity. Mutants of the monoclonal antibody, which was originally isolated from a patient with PBC, were expressed as Fab by phage display, and tested for reactivity against recombinant domains of the E2 subunit. Fab in which the V(H)-encoded portions were reverted to germline lost reactivity against the ILD alone, but recognized a different epitope in a didomain construct encompassing the ILD, hinge region and E1/E3 binding domain. The complete V(H) and V(L )germline revertant was unreactive with the human ILD and didomain, the Escherichia coli didomain, and whole PDC. We hypothesize that the IgM on the surface of the naïve B-cell first recognizes an as yet unidentified antigen, and that accumulation of somatic mutations results in an intermolecular epitope shift directed towards an epitope involving the E1/E3 binding domain. Further mutations result in the specificity being redirected to the ILD. These findings also suggest that bacterial molecular mimicry is not involved in initiating disease.

Immune function, mutant frequency, and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy.

There is evidence for defective DNA repair in xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy, but for increased cancer risk only in xeroderma pigmentosum. Natural and adaptive immune surveillance and mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes were studied in five patients with xeroderma pigmentosum, two with Cockayne's syndrome, and one with trichothiodystrophy. Forty-eight-hour cutaneous hypersensitivity responses to recall antigens excluded anergy and circulating CD3+, CD4+, CD8+, and CD16+ cell numbers were within normal limits in all patients tested, as were proliferative lymphocyte responses to PHA, except in the trichothiodystrophy patient. Proliferative responses to recall antigens (PPD, SKSD, and Candida) showed that all patients responded to one or more antigens. Direct natural killer cytotoxicity measured against the human erythromyeloid leukaemia cell line K562 using a 4-h 51Cr release assay was significantly reduced in xeroderma pigmentosum (specific cytotoxicity less than mean +/- SD greater than 17.4 +/- 9.4 per cent, with effector:target cell ratio of 50:1) compared to normal controls (45.8 +/- 17.8), but normal in Cockayne's syndrome and trichothiodystrophy. Generation of lymphokine activated killer cell activity was normal in the two xeroderma pigmentosum lines tested. The mutant frequency in the xeroderma pigmentosum donors was significantly increased (p less than 0.01) and was elevated in the two Cockayne's syndrome donors, taking age into account. No mutants were observed from the single trichothiodystrophy donor. These findings suggest that reduced natural killer cell activity may contribute to the greatly increased susceptibility to skin cancer in xeroderma pigmentosum.

A new method for measuring clustering in suspension between accessory cells and T lymphocytes.

Specifying the molecular basis and clinical significance of cluster formation between antigen-presenting cells and T lymphocytes will be important in many areas of immunology. In this paper we describe a novel and reproducible technique for measuring cluster formation in suspension between purified human blood monocytes and purified autologous T lymphocytes, and its application to determining the effects of recall antigens and mitogen. Blood monocytes and T lymphocytes from eight normal subjects were separately prelabelled with two different carbocyanine dyes prior to co-culture in suspension with or without antigen (PPD, SKSD) or mitogen (PHA). At 24 h the co-cultures were examined for cluster formation by ultraviolet microscopy and flow cytometry. Control experiments showed that the carbocyanine dyes were non-toxic in vitro, that cell labelling was stable for culture periods up to 120 h, and that the two dyes did not leak from cell to cell. By this technique we measured the proportion of monocytes clustering one or more T lymphocytes in the presence and absence of recall antigen or PHA. There was a close correlation between visual and flow cytometric measurement of monocyte: T lymphocyte clustering (p < 0.001) as well as a close relationship between the ability of the two recall antigens to increase the extent of clustering above baseline (p < 0.001). Antigen-increased cluster formation did not correlate with baseline clustering, unlike PHA-increased clustering, which was related to baseline levels (p = 0.02), suggesting the operation of distinct mechanisms. The method is applicable to measuring cell-cell associations in suspension during extended periods of culture, as well as for the study of agents which might modify intercellular adhesion processes.

A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins.

LeuCAM (CD11/CD18) cell-surface antigens are easily upregulated on cell manipulation ex vivo. A procedure for preparing leucocytes, in which human blood is immediately treated ex vivo with buffered formaldehyde and then the erythrocytes and platelets are removed by lysis and differential centrifugation, has been successfully applied to the analysis of LeuCAM antigen expression by flow cytometry. We show that the increased expression of monocyte CD11/CD18, which occurs when mononuclear leucocytes are separated by a standard Lymphoprep density gradient separation, can be avoided if cells are fixed immediately. Following this fixation polymorphs are unable to upregulate CD11/CD18 in response to fMLP stimulation in vitro. The technique produces lymphocyte, polymorph and monocyte populations that can be clearly defined on the basis of forward scatter and side scatter, and preserves the expression of various surface antigens; the percentages of gated lymphocytes expressing CD3, CD4, and CD8 were similar to those obtained using a commercial fixing and lysis solution. The processing does not render cells permeable to antibodies, as evidenced by our failure to stain cells with antibodies to intracellular antigens. We believed the method to be useful for measuring CD11/CD18 expression on blood leucocytes from normal or pathological specimens and to have application to the measurement of other cells surface antigens which may also be upregulated by the separation procedures.

The use of human lymphoid cell line lymphokine preparations (LCL-LK) as working standards in the bioassay of human leucocyte migration inhibition factor (LIF).

The leucocyte migration inhibition test in agarose as described by Clausen (1971) was modified into a statistically designed assay of LIF activity using human polymorphonuclear leucocytes from single blood donors. Individual assays included a laboratory standard of lymphokine with LIF activity prepared from the culture supernatants of the RPMI 1788 human lymphoblastoid cell line (LCL-LK). Analysis of 157 LIF assays revealed simple criteria by which the acceptability of an individual assay could be judged before subjecting it to statistical analysis. The failure of LIF assays to meet these criteria of acceptability was particularly associated with low areas of control polymorph migration in the absence of lymphokine ('spontaneous migration'). We demonstrate that the statistically designed assay permits the measurement, with precision, of LIF activity in units/ml by reference to a working standard of LCL-LK. We illustrate the use of this assay in the measurement of LIF activity generated by tuberculin-stimulated human peripheral blood lymphocytes.

Sensitization of human lymphocytes in vitro. Kinetics and specificity of the response to hemocyanins.

In vitro primary sensitization of human peripheral blood T-cells to hemocyanins was detected during a 12-14 day culture period with antigen (KLH or HCH)-pulsed macrophages. Primed T-cells proliferate in secondary culture (2-3 days) when restimulated with antigen presented by macrophages. The kinetics of primary sensitization and secondary responsiveness are interrelated and are dependent on the antigen doses employed. Antigen-induced proliferation of cells sensitized in vitro is identical to proliferation of T-cells from immunized donors in terms of antigen specificity and the clonal nature of the response to antigen. Through the use of thymidine suicide techniques, distinct populations of cells responding to KLH or HCH can be demonstrated using cells from actively immunized donors or cells that have been sensitized in vitro to both hemocyanins.

Endothelial cell presentation of antigen to human T cells.

Activation of human T cells requires presentation of antigen by Ia (HLA-DR in man) bearing cells of the mononuclear phagocytic series (macrophages, MO, and more recently Langerhans cells, dendritic cells, and vascular endothelial cells. Since T cells must cross endothelial barriers to enter extravascular tissues during immune reactions, we investigated the role of endothelial cells in antigen presentation. Endothelial cells were cultured from human umbilical veins and identified by classic morphology and specific markers (factor VIII related antigen, and so on). Antigen-pulsed endothelial cells were used to present antigen to MO-depleted human T cells; activation was assessed by 3H-thymidine uptake. The HLA-DR compatible endothelial cells were as effective as MO in reconstituting MO-depleted T-cell responses. The endothelial cell reconstituted responses were antigen specific, HLA-DR restricted, and blocked by monoclonal antibodies to HLA-DR framework structures. Moreover, the T-cell responses were clonal with respect to HLA-DR. A monoclonal antibody completely eliminated MO reconstitution of the MO-depleted response without diminution of endothelial cell reconstitution of the same response. Fibroblasts and smooth muscle cells cultured from the same umbilical veins could not reconstitute the MO-depleted T-cell response. These data indicate that endothelial cells play an important and distinctive role in lymphocyte triggering.

Contrast and electrolyte dynamics of the intravenous pyelogram. II. Disparities in serum and urine concentrations of meglumine and anion.

Intravenous or intra-aortic injections of meglumine iothalamate in dogs indicate a slightly different tissue distribution and excretory pattern of the meglumine cation and the iothalamate anion. Differences in serum and urinary concentrations of these ions suggest some cellular penetration of the meglumine anion. In addition, there seems to be some evidence for both tubular absorption and tubular excretion of meglumine as a minor component in the renal excretion of this ion. No significant differences could be demonstrated for either urinary load or concentration when the sodium salts of iothalamate were compared to the methylglucamine salts.

Contrast and electrolyte dynamics of the intravenous pyelogram. I. Urinary pH and volume changes in the canine I. V. P.

Canine peripheral venous or suprarenal aortic injections of sodium or meglumine iothalamate produced a significant swing towards an alkaline urine only in individual dogs injected with meglumine salts. When mean values were compared for the sodium or meglumine groups as a whole, however, no significant differences could be established for pH change or induced diuresis. With both salts, ATPase inhibition could play a role in urinary electrolyte excretion. Carbonic anhydrase inhibition does not seem to play a significant role in urinary pH changes induced by contrast media.

Glucocorticoid-induced elevations of C1-esterase inhibitor: a mechanism for protection against lethal dose range contrast challenge in rabbits.

Rabbits pretreated with methylprednisolone acquired significant protection against an intravenous challenge of meglumine iodipamide. In comparison to controls, the pretreated rabbits showed moderate elevations of Factor XII, and rather striking elevations of C1-esterase inhibitor. Treated rabbits also showed significantly less granulocytosis. It is believed that the protective effect can be ascribed to the modulation of acute phase reactants by increased concentrations of C1-esterase inhibitor.

Activation systems in contrast idiosyncrasy.

Both animal and human data suggest the possibility that the C1 esterase inhibitor may play an important controlling role in contrast media systemic reactions. This critical controlling protein has a major inhibitory effect on C1, kallikrein, activated factor XII of the intrinsic coagulation system, and on plasmin. In addition, it probably has other inhibitory effects not so well documented. Any circumstance that contributes to a continuing activation of the complement, coagulation, kinin, or fibrinolytic systems may result in partial consumption of the inhibitor and predispose the individual to adverse reactions to contrast challenge.

Changes in complement and coagulation factors in a patient suffering a severe anaphylactoid reaction to injected contrast material: some considerations of pathogenesis.

A patient, suffering a severe anaphylactoid reaction to contrast material injected for an intravenous pyelogram, developed a consumption coagulopathy and evidence of complement activation. Precontrast complement values suggested that the patient had been processing complement via the classical pathway, perhaps as a consequence of an earlier protracted Klebsiella infection. Following contrast injection, a precipitous fall in hemolytic complement (CH50) and in the concentration of the C1 esterase inhibitor (C1INH) developed, as well as a diminution in C4 and C3 with the evolution of C3 conversion products. The possible role that these changes might play in the pathogenesis of idiosyncratic reactions to contrast media is considered.

Effect of endotoxemia on contrast media reactions.

Since complement activation is sharply temperature-dependent, we have examined the effects of fever produced by a very small dose of endotoxin on contrast media lethality in rabbits. After the injection of 8.2 ml/kg of 52% methylglucamine iodipamide, a group of rabbits exhibiting an average temperature elevation of 1.5 degrees C had a 100% mortality rate. This was contrasted to a 30% mortality in control rabbits receiving contrast alone, and no mortality in rabbits receiving endotoxin alone. The rabbits with fever and increased mortality exhibited increased activation of serum complement. From this preliminary data it appears that caution should be observed in performing a contrast examination in a patient with endotoxemia and/or a fever.