Rational design of PD-1-CD28 immunostimulatory fusion proteins for CAR T cell therapy.

BACKGROUND: In many situations, the therapeutic efficacy of CAR T cells is limited due to immune suppression and poor persistence. Immunostimulatory fusion protein (IFP) constructs have been advanced as a tool to convert suppressive signals into stimulation and thus promote the persistence of T cells, but no universal IFP design has been established so far. We now took advantage of a PD-1-CD28 IFP as a clinically relevant structure to define key determinants of IFP activity. METHODS: We compared different PD-1-CD28 IFP variants in a human leukemia model to assess the impact of distinctive design choices on CAR T cell performance in vitro and a xenograft mouse model. RESULTS: We observed that IFP constructs that putatively exceed the extracellular length of PD-1 induce T-cell response without CAR target recognition, rendering them unsuitable for tumour-specific therapy. IFP variants with physiological PD-1 length ameliorated CAR T cell effector function and proliferation in response to PD-L1+ tumour cells in vitro and prolonged survival in vivo. Transmembrane or extracellular CD28 domains were found to be replaceable by corresponding PD-1 domains for in vivo efficacy. CONCLUSION: PD-1-CD28 IFP constructs must mimic the physiological interaction of PD-1 with PD-L1 to retain selectivity and mediate CAR-conditional therapeutic activity.

Three novel presenilin 1 mutations marking the wide spectrum of age at onset and clinical patterns in familial Alzheimer's disease.

Presenilin 1 (PSEN1) mutations are the major cause of autosomal dominant Alzheimer's disease (ADAD). Here we report three novel PSEN1 mutations: Ile238_Lys239insIle, Ala246Pro and Ala164Val from patients who manifested in their twenties, forties and seventies, respectively, with variant clinical presentations of dementia. These cases exemplify the tremendous heterogeneity of clinical phenotypes and age of onset associated with PSEN1 mutations. The possibility of ADAD--not previously suspected in two of our patients--should always be considered in neurodegenerative conditions albeit they might neither exhibit the typical clinical picture of Alzheimer's disease nor early onset dementia, which is regarded the primary clinical sign of hereditary neurodegeneration.

Beyond direct killing-novel cellular immunotherapeutic strategies to reshape the tumor microenvironment.

The clinical use of cellular immunotherapies is gaining momentum and the number of approved indications is steadily increasing. One class of cellular therapies-chimeric antigen receptor (CAR)-modified T cells-has achieved impressive results in distinct blood cancer indications. These existing cellular therapies treating blood cancers face significant relapse rates, and their application beyond hematology has been underwhelming, especially in solid oncology. Major reasons for resistance source largely in the tumor microenvironment (TME). The TME in fact functionally suppresses, restricts, and excludes adoptive immune cells, which limits the efficacy of cellular immunotherapies from the onset. Many promising efforts are ongoing to adapt cellular immunotherapies to address these obstacles, with the aim of reshaping the tumor microenvironment to ameliorate function and to achieve superior efficacy against both hematological and solid malignancies.

DYT7 gene locus for cervical dystonia on chromosome 18p is questionable.

BACKGROUND: A locus implicated in autosomal dominant cervical dystonia was assigned to chromosome 18p in 1 large family more than 15 years ago. This locus was designated DYT7. We reanalyzed the family clinically and genetically. METHODS: Clinical reevaluation of all family members was performed. There was Sanger sequencing of candidate genes, SNP array analysis, and exome sequencing in definitely affected family members. RESULTS: Diagnosis of cervical dystonia was definite in 6 family members and possible in 12. Analysis of candidate genes in 18p revealed no alteration in definitely affected patients. There was no disease causing copy number variant in 18p. No potentially disease-causing mutations were detected in 18p by exome sequencing. The CIZ1 gene, mutated in some cases of cervical dystonia, was excluded. CONCLUSIONS: Location of DYT7 on 18p in autosomal dominant cervical dystonia is questionable. We demonstrate genetic heterogeneity of this form of dystonia.

A Severe Dementia Syndrome Caused by Intron Retention and Cryptic Splice Site Activation in STUB1 and Exacerbated by TBP Repeat Expansions.

Heterozygous pathogenic variants in the STIP1 homologous and U-box containing protein 1 (STUB1) gene have been identified as causes of autosomal dominant inherited spinocerebellar ataxia type 48 (SCA48). SCA48 is characterized by an ataxic movement disorder that is often, but not always, accompanied by a cognitive affective syndrome. We report a severe early onset dementia syndrome that mimics frontotemporal dementia and is caused by the intronic splice donor variant c.524+1G>A in STUB1. Impaired splicing was demonstrated by RNA analysis and in minigene assays of mutated and wild-type constructs of STUB1. The most striking consequence of this splicing impairment was retention of intron 3 in STUB1, which led to an in-frame insertion of 63 amino acids (aa) (p.Arg175_Glu176ins63) into the highly conserved coiled-coil domain of its encoded protein, C-terminus of HSP70-interacting protein (CHIP). To a lesser extent, activation of two cryptic splice sites in intron 3 was observed. The almost exclusively used one, c.524+86, was not predicted by in silico programs. Variant c.524+86 caused a frameshift (p.Arg175fs*93) that resulted in a truncated protein and presumably impairs the C-terminal U-box of CHIP, which normally functions as an E3 ubiquitin ligase. The cryptic splice site c.524+99 was rarely used and led to an in-frame insertion of 33 aa (p.Arg175_Glu176ins33) that resulted in disruption of the coiled-coil domain, as has been previously postulated for complete intron 3 retention. We additionally detected repeat expansions in the range of reduced penetrance in the TATA box-binding protein (TBP) gene by excluding other genes associated with dementia syndromes. The repeat expansion was heterozygous in one patient but compound heterozygous in the more severely affected patient. Therefore, we concluded that the observed severe dementia syndrome has a digenic background, making STUB1 and TBP important candidate genes responsible for early onset dementia syndromes.

Maternal uniparental heterodisomy with partial isodisomy of a chromosome 2 carrying a splice acceptor site mutation (IVS9-2A>T) in ALS2 causes infantile-onset ascending spastic paralysis (IAHSP).

Infantile-onset ascending spastic paralysis (OMIM #607225) is a rare autosomal recessive early onset motor neuron disease caused by mutations in the gene ALS2. We report on a splice acceptor site mutation in intron 9 of ALS2 (IVS9-2A>T) in a German patient from nonconsanguineous parents. The mutation results in skipping of exon 10. This causes a frame-shift in exon 11 and a premature stop codon. Analysis of the parental ALS2 gene revealed heterozygosity for the mutation in the mother but not in the father. Therefore, we studied polymorphic markers scattered along chromosome 2 in both parents and the patient and found maternal uniparental disomy in the patient. While homozygosity was observed at several loci of chromosome 2 including ALS2, other loci were heterozygous, i.e., both maternal alleles were present. The findings can be explained by at least four recombination events during maternal meiosis followed by a meiosis I error and postzygotic trisomy rescue or gamete complementation.

Novel homozygous ALS2 nonsense mutation (p.Gln715X) in sibs with infantile-onset ascending spastic paralysis: the first cases from northwestern Europe.

We describe a previously not recognized nonsense mutation in exon 10 of the ALS2 gene in two sibs with infantile-onset ascending spastic paralysis. The mutation predicts chain termination at amino-acid position 715 of the gene product ALSIN (p.Gln715X). The sibs' parents are descendants of a common ancestor who lived in the northern Netherlands during the eighteenth century. This is the first ALS2 mutation detected in northwestern Europeans. The findings emphasize that mutations in ALS2 also need to be considered in patients from northwestern Europe with early-onset spastic paralysis and amyotrophic or primary lateral sclerosis.

Epigenome-wide DNA methylation profiling in Progressive Supranuclear Palsy reveals major changes at DLX1.

Genetic, epigenetic, and environmental factors contribute to the multifactorial disorder progressive supranuclear palsy (PSP). Here, we study epigenetic changes by genome-wide analysis of DNA from postmortem tissue of forebrains of patients and controls and detect significant (P < 0.05) methylation differences at 717 CpG sites in PSP vs. controls. Four-hundred fifty-one of these sites are associated with protein-coding genes. While differential methylation only affects a few sites in most genes, DLX1 is hypermethylated at multiple sites. Expression of an antisense transcript of DLX1, DLX1AS, is reduced in PSP brains. The amount of DLX1 protein is increased in gray matter of PSP forebrains. Pathway analysis suggests that DLX1 influences MAPT-encoded Tau protein. In a cell system, overexpression of DLX1 results in downregulation of MAPT while overexpression of DLX1AS causes upregulation of MAPT. Our observations suggest that altered DLX1 methylation and expression contribute to pathogenesis of PSP by influencing MAPT.

Transthyretin valine-94-alanine, a novel variant associated with late-onset systemic amyloidosis with cardiac involvement.

A 63-year-old Caucasian male, diagnosed with dilated cardiomyopathy in 1993, remained clinically stable for several years. In 2003, a marked increase of N-terminal pro-natriuretic peptide serum level (611 ng/ml to 4926 ng/ml) was observed; left ventricular (LV) septum thickness was 10 mm. In addition, sensorimotor polyneuropathy and autonomic dysfunction occurred. Further progression of heart failure occurred despite unchanged systolic LV function. Endomyocardial biopsy in 2006 revealed transthyretin amyloidosis by Congo red and immunohistochemical staining, as well as Val94Ala substitution by transthyretin gene analysis. Cardiac amyloid deposition was quantified by technetium-99m-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) scintigraphy. Mutational search of the relatives (n = 1) was unremarkable. The transthyretin Val94Ala mutation is characterized by sensorimotor polyneuropathy, autonomic dysfunction, and gastrointestinal and cardiac involvement with amyloid. This mutation is an addition to the growing spectrum of transthyretin mutations with late onset of clinical symptoms, but noteworthy because of progressive, finally disabling disease course. Final clinical assessment of severity of cardiac involvement in the present patient is rendered complex by possible concomitant or preceding idiopathic dilated cardiomyopathy.

Previously unrecognized missense mutation E126K of PSEN2 segregates with early onset Alzheimer's disease in a family.

Mutations in the gene PSEN2 are a rare cause of early onset Alzheimer's disease (EOAD). PSEN2 sequence variants are often only found in one patient and pathogenicity cannot be formally documented. Here we describe a previously unrecognized sequence change (c.376G>A) in PSEN2 in an EOAD patient and her likewise affected mother. This change results in the exchange of amino acid glutamic acid (E) by lysine (K) at position 126 of the protein (p.E126K). Pathogenicity of the mutation is shown by segregation with disease, evolutionary conservation of E126, and in silico analysis of the mutation.

[Perivascular epithelioid cell tumours of the uterus]. / Perivaskulær epiteloidcelletumor i uterus.

Perivascular epithelioid cell tumours (PEComas) are rare tumours with unknown malignant potential. We report a case of a 48-year-old woman, who had had many tumours, initially diagnosed as leiomyomas, removed from her uterus and vagina over 16 years. A recurrent tumour and revision of prior specimens revealed PEComa. As free surgical margins could not be obtained, the woman is regularly controlled by magnetic resonance imaging. So far recurrence has not been detected. This case shows that knowledge of PEComas is important to ensure relevant follow-up as these are low malignant tumours that tend to reoccur.

The epidemiology of CuZn-SOD mutations in Germany: a study of 217 families.

We screened 217 patients from Germany (n = 213), Austria (n = 2) and Switzerland (n = 2) with a positive family history for amyotrophic lateral sclerosis (ALS) for mutations in the copper/zinc superoxide dismutase (SOD1) gene. We found that 13% of the families tested carried mutations. By analyzing inheritance, we detected a clear-cut co-segregation in 5 of the 28 families; however, in two families with an established mutation, co-segregation was absent. In Germany, the R115G mutation is comparatively frequent and exhibits a specific aggressive phenotype. The L144F mutation, which is the most prevalent mutation in the Balkan countries, and the D90A mutation which is the most frequent SOD1 mutation globally, seem to be the second most common disease-causing mutations in Germany.

Genetic microheterogeneity of human transthyretin detected by IEF.

Mutations of the human transthyretin (TTR) gene have attracted medical interest as a cause of amyloidosis. Recently, we have described in detail an electrophoretic procedure with PAGE followed by IEF in urea gradients for the study of the microheterogeneity of TTR monomers (Altland, K., Winter, P., Sauerborn, M. K., Electrophoresis 1999, 20, 1349-1364). In this paper, we present a study on 49 different mutations of TTR including 33 that result in electrically neutral amino acid substitutions. The aims of the investigation were to test the sensitivity of the procedure to detect TTR variants in patients with TTR amyloidosis and their relatives and to identify some common characteristics that could explain the amyloidogenicity of these variants. We found that all tested amyloidogenic mutations could be detected by our method with the exception of those for which the corresponding variant was absent in plasma samples. Most of the electrically neutral amyloidogenic TTR variants had in common a reduced conformational stability of monomers by the activity of protons and urea. For three variants, e.g. TTR-F64L, TTR-I107V and TTR-V122I, the monomers had a conformational stability close to that of normal monomers but we found experimental and structural arguments for a weakening of the monomer-monomer contact. All types of amyloidogenic mutations affected the stability of TTR tetramers.

Novel mutation in the ALS2 gene in juvenile amyotrophic lateral sclerosis.

We present a 32-year-old Turkish male with juvenile amyotrophic lateral sclerosis 2 and a previously unrecognized homozygous deletion in exon 4 of the ALS2 gene (553delA). Disease progression is more rapid than in the ALS2 phenotype cases described to date. The patient's consanguineous parents carry the mutation in the heterozygous state as do his two unaffected brothers.

Polyacrylamide gel electrophoresis followed by sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis for the study of the dimer to monomer transition of human transthyretin.

Familial amyloidotic polyneuropathy (FAP) is caused by mutations which destabilize transthyretin (TTR) and facilitate the aggregation into extracellular amyloid fibrils preferentially in peripheral nerve and heart tissues. Therapeutic and preventive trials for FAP at the plasma TTR level require a careful study of the destabilization of TTR under variable conditions. We have developed a simple double one-dimensional (D1-D) electrophoretic procedure with polyacrylamide gel electrophoresis (PAGE) followed by sodium dodecylsulfate (SDS) gradient PAGE to study the dimer to monomer transition. TTR is first isolated by PAGE from other plasma proteins. The gel strip containing the TTR fraction is incubated in 2% SDS under varying conditions of temperature, buffer composition, pH, and additives like urea and/or a sulfhydryl-reactive agent, followed by SDS-gradient PAGE for the separation of TTR dimers and monomers. We demonstrate that an unidirectional dimer to monomer transition of normal TTR is achieved at 70-80 degrees C in neutral to mild alkaline buffers or at 37 degrees C and slightly acidic pH (6-7). Addition of urea favors the transition into monomers. Amyloidogenic mutations like amyloidogenic TTR (ATTR)-V30M or ATTR-I107V favor the transition into monomers in buffer systems close to the physiological pH of human plasma. We conclude that this finding has to be considered by any hypothesis on ATTR-derived amyloidogenesis.

Sulfite and base for the treatment of familial amyloidotic polyneuropathy: two additive approaches to stabilize the conformation of human amyloidogenic transthyretin.

Recently, we presented evidence that sulfite protects transthyretin (TTR) from normal human individuals and heterozygotes with amyloidogenic TTR mutations against the decay of tetramers into monomers. In this paper we demonstrate a stabilizing effect of sulfite on TTR tetramers from a familial amyloidotic polyneuropathy (FAP) patient homozygous for the most-common amyloidogenic TTR-V30 M mutation. We compare the conformational stability of partially sulfonated TTR from a heterozygote for normal TTR and amyloidogenic TTR-V30 M with the stability of untreated TTR from a compound heterozygote for amyloidogenic TTR-V30 M and TTR-T119 M known to have only minor or no problems of FAP. Using a combination of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) gradient PAGE we demonstrate that TTR dimers containing amyloidogenic TTR mutations decay into monomers at pH<7.4. Increasing the pH by some 0.2 units within physiological ranges, i.e., pH 7.0-7.4, and sulfonation of TTR were observed to have additive inhibitory effects on the transition of dimers into monomers. We conclude that mild acidifying episodes in the interstitial volume of tissues at risk for amyloidosis could contribute to the development of FAP. Early and permanent efforts to counteract acidosis by treatment with base could possibly help to delay the onset of the disease. The intake of sulfite could support these efforts.