Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Polymorphisms in P2X4R and CAMKK2 may affect TNFα production: Implications for a role in HIV-associated sensory neuropathy.

Polymorphisms in P2X4R and CAMKK2 associate with susceptibility to HIV-associated sensory neuropathy (HIV-SN) - a condition likely mediated by TNFα. As single nucleotide polymorphisms (SNPs) and haplotypes of CAMKK2, and a neighbouring gene P2X4R, mark susceptibility to HIV-SN in South Africans living with HIV, we examined the relationship between P2X4R and CAMKK2 genotypes and TNFα production. Peripheral blood mononuclear cells from 129 healthy donors were stimulated with killed Escherichia coli, and concentrations of soluble TNFα were assessed. Their DNA was genotyped for 22 SNPs in P2X4R and CAMKK2. Three SNPs within P2X4R and two SNPs within CAMKK2 influenced concentrations of TNFα, but these SNP did not associate with risk for HIV-SN. This incongruence may reflect differences in P2X4R haplotypes present in Africans and Europeans. However some CAMKK2 haplotypes were found in both populations, so CAMKK2 polymorphisms may impact upon HIV-SN via effects of the protein on pathways other than TNFα.

Novel kinin B1 receptor splice variant and 5'UTR regulatory elements are responsible for cell specific B1 receptor expression.

The kinin B1 receptor (B1R) is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood. We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV) of human B1R gene with a shorter 5'untranslated region. This novel SV is ≈35% less stable than the wild-type (WT) transcript in lung adenocarcinoma cells (H2126), but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE) -1020 to -766 bp in H2126; positive response element (PRE) -766 to -410 bp in 16HBE; -410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells) were found. These findings reveal complex regulation of B1R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.

Bacterial infection elicits heat shock protein 72 release from pleural mesothelial cells.

Heat shock protein 70 (HSP70) has been implicated in infection-related processes and has been found in body fluids during infection. This study aimed to determine whether pleural mesothelial cells release HSP70 in response to bacterial infection in vitro and in mouse models of serosal infection. In addition, the in vitro cytokine effects of the HSP70 isoform, Hsp72, on mesothelial cells were examined. Further, Hsp72 was measured in human pleural effusions and levels compared between non-infectious and infectious patients to determine the diagnostic accuracy of pleural fluid Hsp72 compared to traditional pleural fluid parameters. We showed that mesothelial release of Hsp72 was significantly raised when cells were treated with live and heat-killed Streptococcus pneumoniae. In mice, intraperitoneal injection of S. pneumoniae stimulated a 2-fold increase in Hsp72 levels in peritoneal lavage (p<0.01). Extracellular Hsp72 did not induce or inhibit mediator release from cultured mesothelial cells. Hsp72 levels were significantly higher in effusions of infectious origin compared to non-infectious effusions (p<0.05). The data establish that pleural mesothelial cells can release Hsp72 in response to bacterial infection and levels are raised in infectious pleural effusions. The biological role of HSP70 in pleural infection warrants exploration.

A comparison of covered vs bare expandable stents for the treatment of aortoiliac occlusive disease.

OBJECTIVE: This trial was conducted to determine if covered stents offer a patency advantage over bare-metal stents in the treatment of aortoiliac arterial occlusive disease. METHODS: The Covered Versus Balloon Expandable Stent Trial (COBEST), a prospective, multicenter, randomized controlled trial, was performed involving 168 iliac arteries in 125 patients with severe aortoiliac occlusive disease who were randomly assigned to receive a covered balloon-expandable stent or bare-metal stent. Patient demographic data, clinical signs and symptoms, TransAtlantic Inter-Society Consensus (TASC) classification, and preprocedure and postprocedure ankle-brachial index measurements were recorded. The primary end points included freedom from binary restenosis and stent occlusion of the treated area, as determined by ultrasound imaging or quantitative visual angiography, or both. Postprocedural follow-up was at 1, 6, 12, and 18 months. RESULTS: Aortoiliac lesions treated with a covered stent were significantly more likely to remain free from binary restenosis than those that were treated with a bare-metal stent (hazard ratio [HR], 0.35; 95% confidence interval (CI), 0.15-0.82; P = .02). Freedom from occlusion was also higher in lesions treated with covered stents than in those treated with a bare-metal stent (HR, 0.28; 95% CI, 0.07-1.09); however, this did not reach statistical significance (P = .07). Subgroup analyses demonstrated a significant difference in freedom from binary restenosis for covered stents in TASC C and D lesions compared with a bare stent (HR, 0.136; 95% CI, 0.042-0.442). This difference was not demonstrated for TASC B lesions (HR, 0.748; 95% CI, 0.235-2.386). CONCLUSIONS: COBEST demonstrates covered and bare-metal stents produce similar and acceptable results for TASC B lesions. However, covered stents perform better for TASC C and D lesions than bare stents in longer-term patency and clinical outcome.

Pathogenic bacteria and TNF do not induce production of macrophage migration inhibitory factor (MIF) by human monocytes.

Elevated serum macrophage migration inhibitory factor (MIF) is associated with severe sepsis, but it is not clear whether bacteria stimulate synthesis of MIF by blood leukocytes directly or via induction of TNF. Here we assess production of MIF mRNA and protein by blood leukocytes from healthy human subjects (n=28) following exposure to bacteria commonly associated with sepsis (Escherichia coli and Streptococcus pneumoniae). Bacteria did not increase levels of MIF mRNA or secreted protein. CD14(+) monocytes were the main cell type producing MIF before and after stimulation. Exposure of leukocytes to TNF did not induce MIF. Hence elevated levels of serum MIF observed in sepsis may not reflect MIF produced by blood leukocytes stimulated directly by bacteria or TNF.

Endotoxin induced TNF and IL-10 mRNA production is higher in male than female donors: correlation with elevated expression of TLR4.

Modification of cytokine production by gender hormones has been postulated to affect disease susceptibility and outcome. Here we investigate the effect of gender and the menstrual cycle on production of cytokines. Mononuclear cells were isolated every week for 10 consecutive weeks from healthy pre-menopausal women and men. TNF and IL-10 mRNA and protein levels were measured as well as membrane CD14 and intracellular TLR4 protein. Endotoxin stimulation of mononuclear cells from men produced more TNF and IL10 mRNA than cells from women. TLR4 expression was also significantly higher in cells from men. These gender differences in the immune response may help to elucidate the sexual dimorphism observed in infectious diseases.