Pattern of time-dependent reduction of histologically determined infarct volume after focal ischaemia in mice

The mouse model of transcranial permanent occlusion of the middle cerebral artery (tpMCAO) is widely used in stroke research. Here we quantified infarct size using a conventional histological method at several post-ischaemic times, going beyond the commonly analysed period of up to 2 days, following artery occlusion. Two different mouse strains, which are widely used for pharmacological studies of neuroprotection and for genetic engineering, were used. A drill whole was made into the skull of anaesthetised mice and ischaemia was induced by electrocoagulation of the middle cerebral artery. In both mouse strains tested (C57Black/6 and NMRI), the measured infarct volumes decreased significantly during the first days after tpMCAO. Notably, 13 days after surgery, ischaemic and sham-operated animals had indistinguishably small lesions, which where in the range of only 5% of the infarct size on day 2 post-ischaemia. The standard method of calculating oedema and shrinkage correction provided no sufficient explanation for this significant decrease in infarct volume. There was, however, evidence that structural changes in the residual ipsilateral hemisphere may compromise the significance of results arising from the method of calculating oedema and shrinkage correction. In conclusion, our study indicates that the pronounced and fast, time-dependent decrease in histologically defined infarct volume can compromise results when studying the lasting neuroprotective effects of potential drugs.     

Screening of blood donors for human cytomegalovirus (HCMV) IgG antibody with an enzyme immunoassay using recombinant antigens.

BACKGROUND: Screening of blood donors for human cytomegalovirus (HCMV) infection is usually performed by the combined detection of specific IgG and IgM antibody. However, in most of the cases of primary infection HCMV IgG seroconversion is observed concomitantly to IgM production and HCMV IgM antibody detection for blood donor screening is subject to a relatively high frequency of false positive results. OBJECTIVE: In the present study a newly established HCMV IgG ELISA based on recombinant antigens (anti-HCMV recombinant IgG ELISA, Biotest) was evaluated in terms of sensitivity and specificity for blood donor screening. STUDY DESIGN: A total of 442 serum samples including follow-up sera of five patients suffering from primary HCMV infection, selected seropositive and seronegative blood donors and routine specimens were comparatively investigated with three HCMV antibody ELISAs (anti-HCMV recombinant IgG ELISA, Biotest; Enzygnost anti-CMV/IgG + IgM, Dade Behring; and Captia CMV-TA, Centocor). RESULTS: IgG seroconversion was detected with anti-HCMV recombinant IgG ELISA as early as IgM in all five patients suffering from primary infection. The alternative ELISAs were less sensitive, detecting seroconversion one to three bleeds later in 2 (Enzygnost anti-CMV/IgG + IgM) and 4 patients (Captia CMV-TA), respectively. Anti-HCMV recombinant IgG ELISA showed a 99.1% agreement with Enzygnost anti-CMV/IgG + IgM and/or Western blot in the preselected blood donors and routine specimens. Relatively high numbers of false negative (n=20) and positive results (n=7) were obtained with Captia CMV-TA. CONCLUSIONS: Our preliminary data suggest that HCMV antibody screening of blood donors can be performed reliably by detection of specific IgG provided that a highly sensitive assay system is used.     

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.     

Intrinsic <Emphasis Type="Italic">MYH7</Emphasis> expression regulation contributes to tissue level allelic imbalance in hypertrophic cardiomyopathy

HCM, the most common inherited cardiac disease, is mainly caused by mutations in sarcomeric genes. More than a third of the patients are heterozygous for mutations in the MYH7 gene encoding for the β-myosin heavy chain. In HCM-patients, expression of the mutant and the wildtype allele can be unequal, thus leading to fractions of mutant and wildtype mRNA and protein which deviate from 1:1. This so-called allelic imbalance was detected in whole tissue samples but also in individual cells. There is evidence that the severity of HCM not only depends on the functional effect of the mutation itself, but also on the fraction of mutant protein in the myocardial tissue. Allelic imbalance has been shown to occur in a broad range of genes. Therefore, we aimed to examine whether the MYH7-alleles are intrinsically expressed imbalanced or whether the allelic imbalance is solely associated with the disease. We compared the expression of MYH7-alleles in non-HCM donors and in HCM-patients with different MYH7-missense mutations. In the HCM-patients, we identified imbalanced as well as equal expression of both alleles. Also at the protein level, allelic imbalance was determined. Most interestingly, we also discovered allelic imbalance and balance in non-HCM donors. Our findings therefore strongly indicate that apart from mutation-specific mechanisms, also non-HCM associated allelic-mRNA expression regulation may account for the allelic imbalance of the MYH7 gene in HCM-patients. Since the relative amount of mutant mRNA and protein or the extent of allelic imbalance has been associated with the severity of HCM, individual analysis of the MYH7-allelic expression may provide valuable information for the prognosis of each patient.     

Cytomegalovirus vector expressing RAE‐1γ induces enhanced anti‐tumor capacity of murine CD8+ T cells

Designing CD8⁺ T‐cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE‐1γ as CD8⁺ T cell‐based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE‐1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD‐1. CMV vector expressing RAE‐1γ potentiated expansion of KLRG1⁺ CD8⁺ T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long‐term maintenance of epitope‐specific CD8⁺ T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8⁺ T‐cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8⁺ T‐cell sensitive tumors.     

Genomic array as compared to karyotyping in myelodysplastic syndromes in a prospective clinical trial

Karyotyping is considered as the gold standard in the genetic subclassification of myelodysplastic syndrome (MDS). Oligo/SNP‐based genomic array profiling is a high‐resolution tool that also enables genome wide analysis. We compared karyotyping with oligo/SNP‐based array profiling in 104 MDS patients from the HOVON‐89 study. Oligo/SNP‐array identified all cytogenetically defined genomic lesions, except for subclones in two cases and balanced translocations in three cases. Conversely, oligo/SNP‐based genomic array profiling had a higher success rate, showing 55 abnormal cases, while an abnormal karyotype was found in only 35 patients. In nine patients whose karyotyping was unsuccessful because of insufficient metaphases or failure, oligo/SNP‐based array analysis was successful. Based on cytogenetic visible abnormalities as identified by oligo/SNP‐based genomic array prognostic scores based on IPSS/‐R were assigned. These prognostic scores were identical to the IPSS/‐R scores as obtained with karyotyping in 95%‐96% of the patients. In addition to the detection of cytogenetically defined lesions, oligo/SNP‐based genomic profiling identified focal copy number abnormalities or regions of copy neutral loss of heterozygosity that were out of the scope of karyotyping and fluorescence in situ hybridization. Of interest, in 26 patients we demonstrated such cytogenetic invisible abnormalities. These abnormalities often involved regions that are recurrently affected in hematological malignancies, and may therefore be of clinical relevance. Our findings indicate that oligo/SNP‐based genomic array can be used to identify the vast majority of recurrent cytogenetic abnormalities in MDS. Furthermore, oligo/SNP‐based array profiling yields additional genetic abnormalities that may be of clinical importance.     

Imaging of Toxocara canis larvae labelled by CFSE in BALB/c mice

Mice are used most often as a model for human toxocarosis caused by Toxocara canis larvae. Variety of symptoms developing during the infection reflects behaviour of the larvae, which are able to escape from the intestine and further invade and damage various host organs. In order to find an approach enabling observation on parasite behaviour in mouse in vivo, we used an epifluorescence method and a small animal imaging system (SAIS). Larvae of T. canis were labelled by carboxyfluorescein succinimidyl ester (CFSE) which incorporated on the parasite gastrointestinal tract. Following infection of BALB/c mice by CFSE-labelled larvae it has been observed that staining had no influence on viability and further migratory activity of the parasites through the host organs (the intestine, liver, lungs and brain) where they were detected by SAIS until day 17 p.i. In addition, the dye did not affect larval antigenic activity as well as the development of related immune response. Imaging of parasites labelled by CFSE, therefore, may represent a promising way to study behaviour of T. canis larvae in a paratenic host.     

QRICH1 variants in Ververi‐Brady syndrome—delineation of the genotypic and phenotypic spectrum

Ververi‐Brady syndrome (VBS, # 617982) is a rare developmental disorder, and loss‐of‐function variants in QRICH1 were implicated in its etiology. Furthermore, a recognizable phenotype was proposed comprising delayed speech, learning difficulties and dysmorphic signs. Here, we present four unrelated individuals with one known nonsense variant (c.1954C > T; p.[Arg652*]) and three novel de novo QRICH1 variants, respectively. These included two frameshift mutations (c.832_833del; p.(Ser278Leufs*25), c.1812_1813delTG; p.(Glu605Glyfs*25)) and interestingly one missense mutation (c.2207G > A; p.[Ser736Asn]), expanding the mutational spectrum. Enlargement of the cohort by these four individuals contributes to the delineation of the VBS phenotype and suggests expressive speech delay, moderate motor delay, learning difficulties/mild ID, mild microcephaly, short stature and notable social behavior deficits as clinical hallmarks. In addition, one patient presented with nephroblastoma. The possible involvement of QRICH1 in pediatric cancer assumes careful surveillance a key priority for outcome of these patients. Further research and enlargement of cohorts are warranted to learn about the genetic architecture and the phenotypic spectrum in more detail.