Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3‐kinase δ (PI3K δ ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain‐of‐function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.     

5-type HPV mRNA versus 14-type HPV DNA test: test performance,over-diagnosis and overtreatment in triage of women with minor cervical lesions

Background

Repeat cytology and HPV testing is used in triage of women with minor cytological lesions. The objective of this study was to evaluate 14-type HPV DNA and 5-type HPV mRNA testing in delayed triage of women with ASC-US/LSIL.

Methods

We compared a DNA test (Roche Cobas 4800) and an 5-type mRNA test (PreTect HPV-Proofer). In total 564 women were included in the study.

Results

The sensitivity among solved cases for CIN3+ were 100 % (15/15) for both tests. The sensitivity for CIN2+ of the HPV DNA test was 100 % (38/38) relative to 79 % (30/38) for the 5-type HPV mRNA test. The corresponding estimates of specificity for CIN2+ among solved cases were 84 % (393/466; 95 % CI: 81–88) and 91 % (451/498; 95 % CI: 88–93). The positive predictive values for CIN3+ were 13.5 % (15/111) for DNA+ and 19.5 % (15/77) for 5-type mRNA+. Significantly more women screened with 5-type mRNA than DNA returned to screening (81 % vs 71 %, p?<?0.01). Subsequently, significantly fewer women were referred for colposcopy/biopsies/treatment (19 % (105/564) vs 29 % (165/564), p?<?0.01).

Conclusions

5-type HPV mRNA is more specific than 14-type HPV DNA in delayed triage of women with ASC-US/LSIL. The referral rate for colposcopy was 57 % higher for DNA+ relative to mRNA+ cases (165 vs 105), with the same detection rate of CIN3+, but the 5-type mRNA test had lower sensitivity for CIN2+. It is important to consider the trade-off between sensitivity and specificity of the diagnostic test when designing screening algorithms.

     

Genetic linkage between erm(B) and vanA in Enterococcus hirae of poultry origin

Vancomycin-resistant enterococci (VRE) have frequently been isolated from Norwegian poultry production following the prohibition of the glycopeptide growth promoter avoparcin since 1995. In the present study, a close genetic linkage between the vanA and erm(B) determinants in an Enterococcus hirae isolate of poultry origin is demonstrated, a result that indicates a mechanism for co-selection and maintenance of vancomycin resistance in absence of selective pressure from avoparcin. A total of 36 vanA-positive enterococci of poultry origin, also phenotypically resistant to erythromycin and/or tetracycline, were analyzed by PCR for identification of erm and tet resistance determinants. An E. hirae isolate harbored erm(B) and tet(K), and in this isolate vanA and erm(B) were located on a BamHI fragment of an approximately 50-kb plasmid. Approximately 3 kb of this fragment was amplified by PCR with vanA and erm(B) primers. Sequence analysis of the region between erm(B) and vanZ of Tn1546 showed a truncated IS1216V inserted downstream of the erm(B) stop codon, aligned with a conserved copy of the 3'-inverted terminal repeat of Tn1546. Mating experiments with the E. hirae isolate as donor and E. faecalis JH2-2 as recipient did not result in any transconjugants, indicating that the vanA/erm(B)-carrying plasmid was nonconjugative under the given experimental conditions.     

Effect of micro-roughness produced by TiO2 blasting--tensile testing of bone attachment by using coin-shaped implants

The aim of the present study was to examine bone response to micro-rough titanium implants. Forty coin-shaped implants were divided into eight groups according to their surface roughness. The first group had electropolished surfaces. The surfaces of implant groups 2-8 were blasted with TiO2 particles with incremental grain sizes ranging from 7.5-12.5 to 270-330 microns. Five implants from each group were placed into the cortical bone of the proximal tibia in New Zealand Black rabbits. To avoid bone overgrowth during the retention phase the implants were fitted into tight polytetrafluoroethylene (PTFE) caps leaving only the flat test surface exposed to bone. The healing period was set to 10 weeks, and implants with attached bone were evaluated using a tensile testing machine. In groups 1-7 a significant correlation between the micro-roughness of the implant surfaces and retention strength was observed. Maximum bone bonding was observed with implants blasted with 180-220 microns grain size (group 7). Blasting with larger TiO2 particles appeared to decrease the effect. The findings suggest that the best grain size of TiO2 particles for optimising retention of titanium implants in cortical bone should be in the 180-220 microns range.     

Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases

We have examined the effect of sphingolipids on the chemotaxis of human natural killer (NK) cells. Messenger RNA for Edg-1, Edg-6 and Edg-8 but not Edg-3, are expressed in these cells. Sphingosine 1 phosphate (SPP), dihydro SPP (DHSPP) or the CC chemokine RANTES (CCL5), but not sphingosine induces the chemotaxis of these cells. Pertussis toxin inhibits the chemotaxis induced by these ligands. Permeabilization of NK cells with streptolysin O (SLO) and introduction of blocking antibodies to the heterotrimeric G proteins, showed that Galpha(i2), Galpha(s), Galpha(q/11) or Galpha(13) mediate the chemotaxis of SPP, whereas Galpha(i2), Galpha(o) or Galpha(q/11) mediate the chemotaxis of DHSPP. Galpha(i2), Galpha(o), Galpha(s), Galpha(q/11), Galpha(z) or Galpha(12 )mediates RANTES-induced NK cell chemotaxis. Further analysis showed that phosphoinositide 3 kinase (PI3K) inhibitors wortmannin and LY294002 inhibit NK cell chemotaxis induced by SPP, DHSPP or RANTES. Blocking antibody to PI3Kgamma inhibits the chemotaxis induced by the three ligands, whereas anti-PI3Kbeta was without effect. In contrast, SPP and DHSPP recruit PI3Kbeta isozyme into NK cell membranes, suggesting that although this isoform is not involved in chemotaxis, it is activated by these phospholipids.     

Itraconazole treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis (CF) patients is a potentially fatal inflammatory disease due to the dual-type immune response provoked by the fungal antigens. Despite serious side effects long-term treatment with corticosteroids is often required. Itraconazole has been reported to be a useful steroid-sparing agent. METHODS: In a retrospective follow-up of 21 CF patients from a total of 250 treated once or twice within a five-year study period (1994-98), 9 patients were treated with systemic glucocorticosteroids in combination with itraconazole and 12 patients were treated with itraconazole (200-600 mg/day) as monotherapy. RESULTS: During treatment the percentage of Aspergillus fumigatus (AF)-positive sputum cultures significantly reduced (P < 0.05); precipitating antibodies to AF decreased significantly in all patients (P < 0.05); forced expiratory volume (FEV1) increased to pre-exacerbation level; total IgE levels decreased in 42% of patients on monotherapy and in 56% on combination therapy. Specific IgE (radioallergosorbant; RAST) level decreased in 6 of 21 patients. Eleven patients had transient increased levels of alanine transaminase (ALAT). One patient had isolated increase in alkaline phosphatase and another in aspartate transaminase (ASAT). CONCLUSIONS: High dose itraconazole as monotherapy or in combination with systemic glucocorticosteroids seems effective in CF patients with ABPA. No hepatotoxicity was observed during long-term therapy.     

Effects of sleep deprivation on extracellular serotonin in hippocampus and frontal cortex of the rat

Sleep deprivation improves the mood of depressed patients, but the exact mechanism behind this effect is unclear. An enhancement of serotonergic neurotransmission has been suggested. In this study, we used in vivo microdialysis to monitor extracellular serotonin in the hippocampus and the frontal cortex of rats during an 8 h sleep deprivation period. These brain regions were selected since both have been implicated in depression. The behavioral state of the animal was continuously monitored by polygraphic recordings during the experiment. Sleep deprivation produced a gradual decline in extracellular serotonin levels, both in the hippocampus and in the frontal cortex. In order to investigate whether the reduction in serotonin was due to other factors than sleep deprivation, i.e. time of day effect, another experiment was performed. Here animals were allowed to sleep during most of the recording period. This experiment showed the expected changes in extracellular serotonin levels: consistently higher levels in the awake, non-sleep deprived animals compared to during sleep, but no time of day effect. The reduction in extracellular serotonin during sleep deprivation may suggest that serotonin does not play a major role in the mood-elevating effect of sleep deprivation. However, since 5-HT levels are strongly behavioral state dependent, by eliminating sleep, there may be a net increase in serotonergic neurotransmission during the sleep deprivation period.     

Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.