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In a healthy udder, immune cells from the peripheral bloodstream migrate into mammary tissue in low numbers to provide baseline immune surveillance, without triggering inflammation. In bovine intramammary inflammation, on the other hand, high amounts of leukocytes are recruited, causing severe inflammation. We were interested in leukocyte subpopulations and functional differences between blood- and milk-derived neutrophils from healthy and inflamed udder quarters. In this context, we found a distinct leukocyte subpopulation profile dependent on the health status of mammary gland quarters, with a predominant T cells population in heathy mammary gland quarters and a shift to macrophages and granulocytes in inflammation. Further, we detected divergent expression of major histocompatibility complex class II and interleukin 2 receptor CD25 on the surface of milk- and blood-derived neutrophils, pointing to antigen presentation and immune modulatory properties. Moreover, we observed differences in production of reactive oxygen species, deviant early and late apoptosis and functional changes in these cells, pointing to an altered metabolic phenotype in milk cells dependent on the health status of mammary gland quarters. These findings provide insights into the functional adaptations of neutrophils in different environments, highlighting the importance of metabolic alterations for immune cell function.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4 + cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4 + cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64 Cu-radiolabeled CD4-Nb1 in CD4 + T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by molecular imaging. Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized , including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Considering the need for advanced probes for noninvasive monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal diagnostics. Quantification of OX40 T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64 Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

Crossbreeding in dairy cattle has been used to improve functional traits, milk composition, and efficiency of Holstein herds. The objective of the study was to compare indicators of the metabolic energy balance, nonesterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), glucose, body condition score (BCS) back fat thickness (BFT), as well as milk yield and milk composition of Holstein and Simmental cows, and their crosses from the prepartum period until the 100th day of lactation at the Livestock Center of the Ludwig Maximilians University (Munich, Germany). In total, 164 cows formed five genetic groups according to their theoretic proportion of Holstein and Simmental genes as follows: Holstein (100% Holstein; n = 9), R1-Hol (51–99% Holstein; n = 30), first generation (F1) crossbreds (50% Holstein, 50% Simmental; n = 17), R1-Sim (1–49% Holstein; n = 81) and Simmental (100% Simmental; n = 27). The study took place between April 2018 and August 2019. BCS, BFT blood parameters, such as BHBA, glucose, and NEFA were recorded weekly. A mixed model analysis with fixed effects breed, week (relative to calving), the interaction of breed and week, parity, calving year, calving season, milking season, and the repeated measure effect of cow was used. BCS increased with the Simmental proportion. All genetic groups lost BCS and BFT after calving. Simmental cows showed lower NEFA values. BHBA and glucose did not differ among genetic groups, but they differed depending on the week relative to calving. Simmental and R1-Sim cows showed a smaller effect than the other genetic groups regarding changes in body weight, BCS, or back fat thickness after a period of a negative energy balance after calving. There was no significant difference for milk yield among genetic groups, although Simmental cows showed a lower milk yield after the third week after calving. Generally, Simmental and R1-Simmental cows seemed to deal better with a negative energy balance after calving than purebred Holstein and the other crossbred lines. Based on a positive heterosis effect of 10.06% for energy corrected milk (ECM), the F1, however, was the most efficient crossbred line.

Research has shown that digestion of A1 β-casein (β-CN) affects gastrointestinal motility and opioid activity through the release of the peptide β-casomorphin-7 (β-CM7). In the case of the A2 variant, the cleavage of β-CM7 does not occur or occurs at a very low rate. Therefore, the aim of the study was to compare the effects of milk containing either homozygote A1 or A2 β-CN on health and growth parameters of dairy calves. Forty-seven neonatal calves (24 females, 23 males) of the breeds German Holstein (GH, n = 9), German Simmental (GS, n = 33) and their crossing (GH × GS, n = 5) were used in a 21-day feeding study. Fecal score (FS), respiratory frequency (RF), and rectal body temperature (BT) were recorded daily, whereas body weight was measured at birth and at day 21 to estimate the average daily weight gain (ADG). Additionally, blood was collected from calves three times during the experimental period and, for the first time, the respective plasma samples were analyzed for intact β-CM7. Consumption of A2-milk led to a lower daily milk intake (dMI) (p < 0.05). Furthermore, fecal consistency was softer for calves fed A2-milk (p < 0.05). Although 44% of A2-calves had diarrhea or revealed a tendency towards it (FS ≥ 3), A1-calves had a prevalence of 21%. Calves with a FS of 4 were offered an electrolyte solution and received a dietary food supplement for the stabilization of the fluid and electrolyte balance. Nevertheless, similar ADG and end weights (EW) of calves fed A1- or A2-milk (p > 0.05) indicate that A2-milk may compensate higher diarrhea rates and lower dMI due to the associated higher protein content. This is the first report of intact β-CM7 in plasma of calves fed milk of either A1 or A2 β-CN. Evidence from this study suggests that due to the change in the amino-acid sequence, A2-milk might be able to prevent or, at least, to minimize the cleavage of β-CM7 in calves.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of bovine paratuberculosis, a chronic granulomatous enteritis leading to economic losses and posing a risk to human health due to its zoonotic potential. The pathogen cannot reliably be detected by standard methods, and immunological procedures during the infection are not well understood. Therefore, the aim of our study was to explore host–pathogen interactions in MAP-infected dairy cows and to improve diagnostic tests. Serum proteomics analysis using quantitative label-free LC-MS/MS revealed 60 differentially abundant proteins in MAP-infected dairy cows compared to healthy controls from the same infected herd and 90 differentially abundant proteins in comparison to another control group from an uninfected herd. Pathway enrichment analysis provided new insights into the immune response to MAP and susceptibility to the infection. Furthermore, we found a higher abundance of Cathepsin S (CTSS) in the serum of MAP-infected dairy cows, which is involved in multiple enriched pathways associated with the immune system. Confirmed with Western blotting, we identified CTSS as a potential biomarker for bovine paratuberculosis. This study enabled a better understanding of procedures in the host–pathogen response to MAP and improved detection of paratuberculosis-diseased cattle.

The objective of this study was to phenotype visceral adipose tissue (VAT) in pigs. In this context, the ability to detect VAT by using the DXA CoreScan mode within the enCORE software, version 17 (GE Healthcare) was evaluated in comparison with MRI measurements (Siemens Magnetom C!) of the same body region. A number of 120 crossbred pigs of the F1 and F2 generation, with the parental breeds Large White, Landrace, Piétrain, and Duroc, were examined at an age of 150 days. A whole-body scan in two different modes (“thick”, “standard”) was carried out by a GE Lunar iDXA scanner. Very strong relationships (R2 = 0.95, RMSE = 175 cm3) were found for VAT between the two DXA modes. The comparison of VAT measured by MRI and DXA shows high linear relationships (“thick”: R2 = 0.76, RMSE = 399.25 cm3/“standard”: R2 = 0.71, RMSE = 443.42 cm3), but is biased, according to the Bland–Altman analysis. A variance analysis of VAT shows significant differences for both DXA modes and for MRI between male and female pigs, as well as between F1 and F2. In conclusion, DXA “CoreScan” has the ability to estimate VAT in pigs with a close relationship to MRI but needs bias correction.