Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans

The fungus Cryptococcus neoformans is a major cause of morbidity and mortality in patients with impaired CD4(+) T cell function, particularly those with AIDS. To identify cryptococcal antigens that could serve as vaccine candidates by stimulating T cell responses, C. neoformans-reactive CD4(+) T cell hybridomas were generated by immunization of C57BL/6 mice and fusion of splenocytes with thymoma cells. The antigen that stimulated one of the hybridomas, designated P1D6, to produce IL-2 was purified to homogeneity by sequential anion exchange chromatography, hydrophobic interaction chromatography, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosylation, the antigen of interest was named MP98. MP98 was N terminal-sequenced, and the gene encoding the protein was cloned and sequenced. Recombinant MP98, expressed in Saccharomyces cerevisiae, stimulated P1D6 to produce IL-2. Analysis of the derived 458-aa sequence of MP98 reveals an N-terminal cleavable signal sequence, a polysaccharide deacetylase domain found in fungal chitin deacetylases, and a serine/threonine-rich C-terminal region. Overall, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. Thus, a C. neoformans mannoprotein has been characterized that stimulates T cell responses and has molecular properties of a chitin deacetylase.     

Homozygous and compound heterozygous mutations in the ATP6V1B1 gene in patients with renal tubular acidosis and sensorineural hearing loss

Mohebbi N, Vargas‐Poussou R, Hegemann SCA, Schuknecht B, Kistler AD, Wüthrich RP, Wagner CA. Homozygous and compound heterozygous mutations in the ATP6V1B1 gene in patients with renal tubular acidosis and sensorineural hearing loss. Distal renal tubular acidosis (dRTA) is characterized by the inability to excrete acid in the renal collecting ducts resulting in inappropriately alkaline urine and hyperchloremic (normal anion gap) metabolic acidosis in the context of a normal (or near‐normal) glomerular filtration rate. Inborn dRTA can be due to autosomal dominant or recessive gene defects. Clinical symptoms vary from mild acidosis, incidental detection of kidney stones or renal tract calcification to severe findings such as failure to thrive, severe metabolic acidosis, and nephrocalcinosis. The majority of patients with recessive dRTA present with sensorineural hearing loss (SNHL). Few cases with abnormal widening of the vestibular aqueduct have been described with dRTA. Mutations in three different genes have been identified, namely SLC4A1, ATP6V1B1, and ATP6V0A4. Patients with mutations in the ATP6V1B1 proton pump subunit develop dRTA and in most of the cases sensorineural hearing loss early in childhood. We present two patients from two different and non‐consanguineous families with dRTA and SNHL. Direct sequencing of the ATP6V1B1 gene revealed that one patient harbors two homozygous mutations and the other one is a compound heterozygous. To our knowledge, this is the first case in the literature describing homozygosity in the same dRTA gene on both alleles.     

Glucagon-like peptide-1 receptor agonists as neuroprotective agents for ischemic stroke: a systematic scoping review

Stroke mortality and morbidity is expected to rise. Despite considerable recent advances within acute ischemic stroke treatment, scope remains for development of widely applicable neuroprotective agents. Glucagon-like peptide-1 receptor agonists (GLP-1RAs), originally licensed for the management of Type 2 Diabetes Mellitus, have demonstrated pre-clinical neuroprotective efficacy in a range of neurodegenerative conditions. This systematic scoping review reports the pre-clinical basis of GLP-1RAs as neuroprotective agents in acute ischemic stroke and their translation into clinical trials. We included 35 pre-clinical studies, 11 retrospective database studies, 7 cardiovascular outcome trials and 4 prospective clinical studies. Pre-clinical neuroprotection was demonstrated in normoglycemic models when administration was delayed by up to 24 h following stroke induction. Outcomes included reduced infarct volume, apoptosis, oxidative stress and inflammation alongside increased neurogenesis, angiogenesis and cerebral blood flow. Improved neurological function and a trend towards increased survival were also reported. Cardiovascular outcomes trials reported a significant reduction in stroke incidence with semaglutide and dulaglutide. Retrospective database studies show a trend towards neuroprotection. Prospective interventional clinical trials are on-going, but initial indicators of safety and tolerability are favourable. Ultimately, we propose that repurposing GLP-1RAs is potentially advantageous but appropriately designed trials are needed to determine clinical efficacy and cost-effectiveness.     

Dilution of candidates: the case of iron-related genes in restless legs syndrome

Restless legs syndrome (RLS) is a common multifactorial disease. Some genetic risk factors have been identified. RLS susceptibility also has been related to iron. We therefore asked whether known iron-related genes are candidates for association with RLS and, vice versa, whether known RLS-associated loci influence iron parameters in serum. RLS/control samples (n=954/1814 in the discovery step, 735/736 in replication 1, and 736/735 in replication 2) were tested for association with SNPs located within 4 Mb intervals surrounding each gene from a list of 111 iron-related genes using a discovery threshold of P=5 × 10⁻⁴. Two population cohorts (KORA F3 and F4 with together n=3447) were tested for association of six known RLS loci with iron, ferritin, transferrin, transferrin-saturation, and soluble transferrin receptor. Results were negative. None of the candidate SNPs at the iron-related gene loci was confirmed significantly. An intronic SNP, rs2576036, of KATNAL2 at 18q21.1 was significant in the first (P=0.00085) but not in the second replication step (joint nominal P-value=0.044). Especially, rs1800652 (C282Y) in the HFE gene did not associate with RLS. Moreover, SNPs at the known RLS loci did not significantly affect serum iron parameters in the KORA cohorts. In conclusion, the correlation between RLS and iron parameters in serum may be weaker than assumed. Moreover, in a general power analysis, we show that genetic effects are diluted if they are transmitted via an intermediate trait to an end-phenotype. Sample size formulas are provided for small effect sizes.     

Reproducibility of 18F-FDG PET uptake measurements in head and neck squamous cell carcinoma on both PET/CT and PET/MR

Objective:

To investigate reproducibility of fluorine-18 fludeoxyglucose (¹⁸F-FDG) uptake on ¹⁸F-FDG positron emission tomography (PET)/CT and ¹⁸F-FDG PET/MR scans in patients with head and neck squamous cell carcinoma (HNSCC).

Methods:

30 patients with HNSCC were included in this prospective study. The patients were scanned twice before radiotherapy treatment with both PET/CT and PET/MR. Patients were scanned on the same scanners, 3 days apart and according to the same protocol. Metabolic tumour activity was measured by the maximum and peak standardized uptake value (SUV_max and SUV_peak, respectively), and total lesion glycolysis from the metabolic tumour volume defined from ≥50% SUV_max. Bland–Altman analysis with limits of agreement, coefficient of variation (CV) from the two modalities were performed in order to test the reproducibility. Furthermore, CVs from SUV_max and SUV_peak were compared. The area under the curve from cumulative SUV–volume histograms were measured and tested for reproducibility of the distribution of ¹⁸F-FDG uptake.

Results:

24 patients had two pre-treatment PET/CT scans and 21 patients had two pre-treatment PET/MR scans available for further analyses. Mean difference for SUV_max, peak and mean was approximately 4% for PET/CT and 3% for PET/MR, with 95% limits of agreement less than ±20%. CV was small (5–7%) for both modalities. There was no significant difference in CVs between PET/CT and PET/MR (p = 0.31). SUV_max was not more reproducible than SUV_peak (p = 0.09).

Conclusion:

¹⁸F-FDG uptake in PET/CT and PET/MR is highly reproducible and we found no difference in reproducibility between PET/CT and PET/MR.

Advances in knowledge:

This is the first report to test reproducibility of PET/CT and PET/MR.Functional imaging with fluorine-18 fludeoxyglucose positron emission tomography combined with CT (¹⁸F-FDG PET/CT) has been shown to be useful for prognostication of head and neck squamous cell carcinoma (HNSCC),^1–3 and the use of ¹⁸F-FDG PET/CT has also been shown to reduce interobserver variability in target delineation for radiotherapy.^4,5 Furthermore, ¹⁸F-FDG PET/CT can identify regions of the tumour with a high risk of relapse, leading to the idea that ¹⁸F-FDG uptake might be a target for dose painting.^6,7 Finally, ¹⁸F-FDG PET/CT may be used in response evaluation.^8,9 Maximum standardized uptake value (SUV_max) has for many years been the main uptake measurement in prognostic studies for various malignancies. More recent studies have focused on demonstrating prognostic value of PET/CT-based volumetric parameters such as metabolic tumour volume (MTV) and total lesion glycolysis (TLG). MTV is the sum of the volume of voxels with standard uptake value (SUV) exceeding a certain threshold value in a tumour, and TLG is calculated by multiplying MTV and the mean standardized uptake value (SUV_mean) of the MTV. These volume-based PET parameters have increasingly gained interest and have been reported to be significant prognostic factors for various malignancies including HNSCC.^{10–13 18}F-FDG PET/CT is currently not routinely recommended as a diagnostic tool in HNSCC except in very specific situations,¹⁴ but reproducibility of the ¹⁸F-FDG signal is a prerequisite for a more widespread use of ¹⁸F-FDG PET for the above-mentioned indications. Yet, only a few studies of the reproducibility of ¹⁸F-FDG PET/CT exist^8,15–21 and none of these studies includes patients with HNSCC.MRI is gaining acceptance as an imaging modality for oncology as it offers superior soft-tissue contrast compared with CT alone, and it has been suggested that information from PET/CT and MR is complementary in head and neck cancer.²² The introduction of the integrated PET/MR scanner offers a unique opportunity to combine the high soft-tissue contrast of MR with the functional imaging from PET within a single imaging session. PET/MR is still in its infancy, but the combined modality imaging is potentially useful in the management of patients with HNSCC.^22–28 However, the same criteria of reproducibility as with PET/CT should be upheld by this new modality. The purpose of this prospective test–retest study is to assess the reproducibility of both ¹⁸F-FDG PET/CT and ¹⁸F-FDG PET/MR in a homogenous cohort of patients with HNSCC.     

Storage of platelet concentrates using ion exchange resin charged with dibasic phosphate.

Platelet concentrates were prepared at twice the normal concentration and stored at room temperature for 7 days in either standard bags (controls) or bags to which 1 or 2 g of Amberlite resin beads charged with dibasic phosphate had been added. The resin beads served as a buffer system by providing a "slow release" form of phosphate ions as well as by binding CO2 produced during platelet metabolism. Control platelets demonstrated rapid falls in pH, ATP content, morphology score, and thrombin-induced nucleotide release after 24 hr of storage with a fall in pH to less than 6.0 by day 3. Profound ultrastructural changes and a rise in pO2, suggesting loss of platelet viability, accompanied these changes. In contrast, the resin-stored platelets remained near normal after 24 hr of storage, with preservation of discoid morphology, 95% of ATP levels, excellent ultrastructural appearance, and evidence of continued oxygen consumption after 3 days of storage. Even after 7 days of storage, ATP levels remained greater than 50% of baseline and ultrastructurally intact platelets were seen. In the 1-g resin bags the pH remained at baseline levels (6.9-7.0), while there was a rise in pH in the 2-g resin bags. These results demonstrate the beneficial effects of maintaining a higher pH during platelet storage and provide a new approach to studying the metabolic changes that occur during longer term storage.     

B-cell lymphoma after angioimmunoblastic lymphadenopathy: a case with oligoclonal gene rearrangements associated with Epstein-Barr virus

We describe a patient with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), who subsequently developed large-cell immunoblastic lymphoma of B-cell immunophenotype. At the time of the initial diagnosis, histologic examination of an inguinal lymph node showed typical features of AILD, and there was no evidence of a monoclonal B-cell population by immunohistochemical analysis. In situ hybridization and Southern blot analysis for Epstein-Barr virus (EBV) were negative. At autopsy 2 years later, the patient had widespread lymph node and organ involvement by large-cell immunoblastic lymphoma of B-cell immunophenotype. Southern blot analysis performed on DNA extracted from lymph nodes, liver, and spleen showed two patterns of Ig heavy chain and kappa light chain gene rearrangements. The T-cell receptor beta chain gene was in the germline configuration. Analysis with an EBV terminal repeat region probe showed two clonal populations that paralleled the Ig gene rearrangement studies. Double-labeling immunohistochemistry and in situ hybridization confirmed the presence of EBV within the neoplastic B cells. The data support the hypothesis that EBV was not etiologically related to AILD in this case, and that EBV proliferation may occur after the onset of the disease. Further, the data suggest that some B-cell lymphomas that arise in the setting of AILD resemble EBV-associated B-cell lymphomas that arise in other immunodeficiency states.     

Effect of n-3 and n-6 fatty acids on proliferation and differentiation of promyelocytic leukemic HL-60 cells

Promyelocytic leukemic HL-60 cells were incubated with different fatty acids. Arachidonic acid (AA; 20:4, n-6) and eicosapentaenoic acid (EPA; 20:5, n-3) were the most potent inhibitors of proliferation in a dose- dependent way. Retinoic acid (RA) was used as a positive control. Inhibitors of cyclooxygenase and lipoxygenase or addition of antioxidants did not influence the effect of EPA or AA on cell proliferation. Increased capacity to generate superoxide anions after phorbol ester treatment and a reduced serglycin messenger RNA level in cells treated with AA or EPA indicated that these fatty acids induced differentiation in HL-60 cells similar to that induced by RA. However, down-regulation of the c-myc mRNA level, also typical for differentiation with RA in HL-60 cells, was not observed in cells incubated with AA or EPA. Flow cytometric analyses showed that in cultures incubated with AA or EPA, the proportion of cells in the G1 phase of the cell cycle increased. Similar effects were observed with RA. By flow cytometry and light scatter analyses it could be shown that AA made 8% of the cells apoptotic and 7% necrotic. The corresponding numbers were 21% and 10% for RA-treated cells, and 19% and 32% for EPA- treated cells. The present study shows that AA and EPA reduce the proliferation rate of HL-60 cells. This is mediated by mechanisms independent of eicosanoids or lipid peroxidation products and is due to effects both on apoptosis/necrosis and cell differentiation.     

Treatment of acute lymphoblastic leukemia in adolescents and a-dults: A retrospective study of 41 patients (1970-1973)

During the period from January 1970 until December 1973, therapy was started in 41 previously untreated adolescents and adults with acute lymphoblastic leukemia. Induction therapy was started with vincristine and prednisone in all patients, resulting in complete remission in 19 and death due to infection during the first month in one case. After 3 wk on these two drugs, the addition of daunorubicin was required in the remaining 21 patients. Fifteen of these obtained remission, one died during induction therapy, and five patients were unresponsive to this therapy, as well as to all subsequent induction schemes. The overall remission rate was 83%. Significantly higher initial leukocyte counts were found in the group treated with vincristine, prednisone, and daunorubicin. Meningeal leukemia prophylaxis, by either periodic methotrexate injections given intrathecally or a combination of cranial irradiation and intrathecally administrated methotrexate, was administered in 29 therapy responders. The median duration of complete remission obtained with various maintenance therapy schemes was 13 mo. No differences were seen in the results obtained in patients between 14 and 20 yr of age and older patients. Twenty-two patients relapsed within 2-37 mo. Relapses were confined to the central nervous system in two cases, to the bone marrow in 18, and to the bone marrow and CNS simultaneously in two. A second remission was obtained in 17 cases (77%). The median survival time of the whole group was 27 mo, as compared with 32 mo for therapy responders and 7 mo for the nonresponders. The percentage and duration of remission and the survival time in our group of adolescents and adults were comparable to those currently being achieved in other centers, but not as good as those reported for children treated with the same protocols.     

Persistent reversed end diastolic flow in the fetal middle cerebral artery: an ominous finding

Fetal persistent middle cerebral artery reversed end diastolic flow is a rare and ominous finding. Previous cases have been associated with intracranial hemorrhage, growth restriction, anaemia, and hepatic anomaly. Intrauterine demise or early neonatal death is a common outcome. We report the case of persistent middle cerebral artery reversed end diastolic flow in a well-grown fetus at 32 weeks’ gestation resulting from acute, severe anaemia due to a large feto-maternal hemorrhage. An emergency cesarean section was performed and the neonate required advanced resuscitation and immediate blood transfusion. Postnatal magnetic resonance imaging confirmed a hemorrhagic parietal infarct and bilateral ischaemic changes in the basal ganglia. This provides further evidence that persistent middle cerebral artery reversed end diastolic flow in any fetus is an ominous finding warranting urgent diagnostic evaluation and/or delivery.