Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA''s mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt''s lymphoma cell lines. Phagocytosis contributed to DARA''s anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.     

Rating of perceived exertion as a tool for prescribing and self regulating interval training: a pilot study

The aim of the present study was to analyse the usefulness of the 6-20 rating of perceived exertion (RPE) scale for prescribing and self-regulating high-intensity interval training (HIT) in young individuals. Eight healthy young subjects (age = 27.5±6.7 years) performed maximal graded exercise testing to determine their maximal and reserve heart rate (HR). Subjects then performed two HIT sessions (20 min on a treadmill) prescribed and regulated by their HR (HR: 1 min at 50% alternated with 1 min at 85% of reserve HR) or RPE (RPE: 1 minute at the 9-11 level [very light-fairly light] alternated with 1 minute at the 15-17 level [hard-very hard]) in random order. HR response and walking/running speed during the 20 min of exercise were compared between sessions. No significant difference between sessions was observed in HR during low- (HR: 135±15 bpm; RPE: 138±20 bpm) and high-intensity intervals (HR: 168±15 bpm; RPE: 170±18 bpm). Walking/running speed during low- (HR: 5.7±1.2 km · h⁻¹; RPE: 5.7±1.3 km · h⁻¹) and high-intensity intervals (HR: 7.8±1.9 km · h⁻¹; RPE: 8.2±1.7 km · h⁻¹) was also not different between sessions. No significant differences were observed in HR response and walking/running speed between HIT sessions prescribed and regulated by HR or RPE. This finding suggests that the 6-20 RPE scale may be a useful tool for prescribing and self-regulating HIT in young subjects.     

Evidence for the Presence of a Ganglioside Transfer Protein in Brain

An extract from rat brain has been shown to catalyze the transfer of ganglioside GM1 from sonicated vesicles to erythrocyte ghosts. It also enhanced the transfer of GM1 to a crude neuronal membrane preparation, whereas myelin took up only a very limited amount. The transfer activity was heat-labile. Similar transfer activities were found in extracts from bovine gray and white matter, that of the former being comparable to rat brain whereas the latter was greater per milligram protein.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Transmission potential,skin inflammatory response,and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background

Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.

Results

The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).

Conclusion

Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

     

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice

Background

Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results

Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion

A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users.     

A class of Bayesian methods to combine large numbers of genotyped and non-genotyped animals for whole-genome analyses

Background

To obtain predictions that are not biased by selection, the conditional mean of the breeding values must be computed given the data that were used for selection. When single nucleotide polymorphism (SNP) effects have a normal distribution, it can be argued that single-step best linear unbiased prediction (SS-BLUP) yields a conditional mean of the breeding values. Obtaining SS-BLUP, however, requires computing the inverse of the dense matrix G of genomic relationships, which will become infeasible as the number of genotyped animals increases. Also, computing G requires the frequencies of SNP alleles in the founders, which are not available in most situations. Furthermore, SS-BLUP is expected to perform poorly relative to variable selection models such as BayesB and BayesC as marker densities increase.

Methods

A strategy is presented for Bayesian regression models (SSBR) that combines all available data from genotyped and non-genotyped animals, as in SS-BLUP, but accommodates a wider class of models. Our strategy uses imputed marker covariates for animals that are not genotyped, together with an appropriate residual genetic effect to accommodate deviations between true and imputed genotypes. Under normality, one formulation of SSBR yields results identical to SS-BLUP, but does not require computing G or its inverse and provides richer inferences. At present, Bayesian regression analyses are used with a few thousand genotyped individuals. However, when SSBR is applied to all animals in a breeding program, there will be a 100 to 200-fold increase in the number of animals and an associated 100 to 200-fold increase in computing time. Parallel computing strategies can be used to reduce computing time. In one such strategy, a 58-fold speedup was achieved using 120 cores.

Discussion

In SSBR and SS-BLUP, phenotype, genotype and pedigree information are combined in a single-step. Unlike SS-BLUP, SSBR is not limited to normally distributed marker effects; it can be used when marker effects have a t distribution, as in BayesA, or mixture distributions, as in BayesB or BayesC π. Furthermore, it has the advantage that matrix inversion is not required. We have investigated parallel computing to speedup SSBR analyses so they can be used for routine applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1297-9686-46-50) contains supplementary material, which is available to authorized users.     

Xenogeneic transplantation of articular chondrocytes into full-thickness articular cartilage defects in minipigs: fate of cells and the role of macrophages

Xenogeneic or allogeneic chondrocytes hold great potential to build up new cartilage in vivo. However, immune rejection is a major concern for the utility of universal donor-derived cells. In order to verify the reported immune privilege of chondrocytes in vivo, the aim of this study was to assess engraftment of human articular chondrocytes (HAC) in minipig knee cartilage defects and their contribution to cartilage regeneration. HAC were transplanted matrix-assisted within two hydrogels into full-thickness cartilage defects of minipigs or implanted ectopically into immune deficient mice to assess redifferentiation capacity. At 2 and 4 weeks after surgery, cell-persistence and host cell invasion were monitored by species-specific in situ hybridization and RT-PCR. Early tissue regeneration was evaluated by histomorphometry and a modified O’Driscoll score. HAC capable of successful in vivo chondrogenic redifferentiation persisted at ectopic sites for 4 weeks in both carrier materials. Early defect regeneration involved extensive host cell invasion and a decline of HAC to less than 5 % of initial cell numbers in 6/12 defects within 2 weeks. Few clusters of persisting HAC within collagen type II-rich tissue were surrounded by porcine macrophages. Four weeks after cell transplantation, most of the defects contained well-integrated cell-rich tissue free of human cells with no apparent difference between hydrogel carriers. In summary, HAC failed to engraft in porcine articular cartilage defects despite their ability for successful in vivo redifferentiation. The co-localization of macrophages to hydrogel-implanted HAC suggests active graft rejection without evidence for an immune-privileged status of xenogeneic chondrocytes in a large animal joint.     

The challenge for genetic epidemiologists: how to analyze large numbers of SNPs in relation to complex diseases

Background

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.

Results

Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.

Conclusion

The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.