Effects of sample matrix in the measurement of antithrombin by LC-MS: A role for immunocapture.

Introduction: The sample matrix composition, which is greatly affected by the type of blood collection tube used during phlebotomy, is of major importance in laboratory testing as it can influence test results. We developed an LC-MRM-MS test to molecularly characterize antithrombin in citrate plasma. The test principle differs greatly from traditional laboratory tests and the influence of varying plasma sample matrices is largely unknown. Objectives: To identify whether variations in sample matrix affect the LC-MRM-MS test for antithrombin and assess whether sample pre-processing by immunocapture reduces matrix-specific effects. Methods: Samples (n = 45) originating from four different blood collection tubes (sodium citrate, lithium heparin, K2-EDTA and K2-EDTA with protease inhibitors) were processed directly or after immunocapture. Antithrombin was digested into proteotypic peptides, which were monitored by LC-MRM-MS. Results from lithium heparin and the K2-EDTA matrices were compared to the standard sample matrix, sodium citrate, using Deming regression analysis and repeated measures one-way ANOVA. Results: Deming regression analysis of directly processed samples revealed slopes deviating >5% from the line of identity for at least six out of 22 peptides in all matrices. Significant differences between all matrices were found upon analysis by ANOVA for at least 10 peptides. Pre-processing by immunocapture led to slopes within 5% of the line of identity for nearly all peptides of the matrices. Furthermore, significant differences between matrices after immunocapture were only observed for four peptides. Conclusion: Variations in the sample matrix affect the measurement of antithrombin by LC-MRM-MS, but observed effects are greatly reduced upon pre-processing by immunocapture.

Tbet-positive regulatory T cells accumulate in oropharyngeal cancers with ongoing tumor-specific type 1 T cell responses.

Regulatory T cells (Tregs) may comprise different subsets allowing them to efficiently suppress different types of effector T cells. In this study, we show that high numbers of both conventional and Tbet co-expressing Foxp3hi Tregs accumulate in human papilloma virus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC). The infiltration of Tbet+ Foxp3+ Tregs was strongly correlated with a concomitant tumor-specific and conventional type 1-oriented intratumoral T cell infiltrate. Both conventional CD4+CD25+CD127-Foxp3hi Tregs and their Tbethi counterparts exhibited an activated phenotype, co-expressed high levels of CTLA4 and Helios and exhibited a maximally demethylated Foxp3 gene locus TSDR, indicating their full capacity to impede a type 1 effector T cell response. Interestingly, while the prognostic value of conventional Tregs was neutral, a high intratumoral frequency of Tbet+ Tregs was associated with prolonged disease-specific survival, most likely because their presence reflected high numbers of effector T cells. The presence of these Tbet+ Tregs may in part explain why a dense type 1-oriented immune infiltrate in OPSCC is not enough to fully control tumor growth.

The blood mMDSC to DC ratio is a sensitive and easy to assess independent predictive factor for epithelial ovarian cancer survival.

Epithelial ovarian cancer (EOC) may cause abnormal blood levels of leukocytes. This paraneoplastic manifestation is associated with a worse response to therapy and shorter survival. To understand the complexity and nature of these leukocytes, we dissected the different populations of myeloid cells and analyzed their relation to clinical outcome. Therefore, baseline blood samples of 36 EOC patients treated either with carboplatin/doxorubucin or with gemcitabine were analyzed for different subsets of monocytes/macrophages, myeloid derived suppressor cells (MDSC) and dendritic cells (DC) using multiparameter flow cytometry as well as functional assays for myeloid cell mediated suppression of antigen-specific T cell reactivity. Healthy donor blood served as control. EOC patients displayed an increase in monocytes/macrophages, monocytic MDSC (mMDSC) and CD33-CD11b+CD14-CD15- double-negative MDSC (CD33- dnMDSC) and a decrease in the frequency of DC, across all EOC subtypes. A low frequency of DC and high frequencies of monocytes/macrophages and mMDSC, but not CD33- dnMDSC, were associated with poor overall survival. Patient's monocytes/macrophages and mMDSC, but not CD33- dnMDSC, were shown to suppress T cell reactivity in vitro. The mMDSC and DC frequencies were not altered upon treatment. Importantly, the mMDSC to DC ratio was the strongest independent, highly sensitive and specific, predictive factor for survival. This was irrespective of the type of chemotherapy or disease stage and outperformed classical parameters as WHO status or time from last chemotherapy. Thus, the baseline blood mMDSC to DC ratio is a robust, independent and easy to analyze predictive factor for EOC survival, and may assist patient selection for immunotherapy.

Impact of (chemo)radiotherapy on immune cell composition and function in cervical cancer patients.

New treatments based on combinations of standard therapeutic modalities and immunotherapy are of potential use, but require a profound understanding of immune modulatory properties of standard therapies. Here, the impact of standard (chemo)radiotherapy on the immune system of cervical cancer patients was evaluated. Thirty patients with cervical cancer were treated with external beam radiation therapy (EBRT), using conventional three-dimensional or intensity modulated radiation therapy without constraints for bone marrow sparing. Serial blood sampling for immunomonitoring was performed before, midway and at 3, 6 and 9 weeks after EBRT to analyze the composition of lymphocyte and myeloid-cell populations, the expression of co-stimulatory molecules, T-cell reactivity and antigen presenting cell (APC) function. Therapy significantly decreased the absolute numbers of circulating leukocytes and lymphocytes. Furthermore, the capacity of the remaining T cells to respond to antigenic or mitogenic stimulation was impaired. During treatment the frequency of both CD4+ and CD8+ T cells dropped and CD4+ T cells displayed an increased expression of programmed cell death-1 (PD-1). In vitro blocking of PD-1 successfully increased T-cell reactivity in all five samples isolated before radiotherapy but was less successful in restoring reactivity in samples isolated at later time points. Moreover, (chemo)radiotherapy was associated with an increase in both circulating monocytes and myeloid-derived suppressor cells (MDSCs) and an impaired capacity of APCs to stimulate allogeneic T cells. T-cell reactivity was slowly restored at 6-9 weeks after cessation of therapy. We conclude that conventional (chemo)radiotherapy profoundly suppresses the immune system in cervical cancer patients, and may restrict its combination with immunotherapy.

Tumor-infiltrating CD14-positive myeloid cells and CD8-positive T-cells prolong survival in patients with cervical carcinoma.

One of the hallmarks of cancer is the influx of myeloid cells. In our study, we investigated the constitution of tumor-infiltrating myeloid cells and their relationship to other tumor-infiltrating immune cells, tumor characteristics and the disease-specific survival of patients with cervical cancer (CxCa). Triple-color immunofluorescence confocal microscopy was used to locate, identify and quantify macrophages (CD14), their maturation status (CD33) and their polarization (CD163) in a cohort of 86 patients with cervical carcinoma. Quantification of the numbers of myeloid cells revealed that a strong intraepithelial infiltration of CD14+ cells, and more specifically the population of CD14+CD33-CD163- matured M1 macrophages, is associated with a large influx of intraepithelial T lymphocytes (p = 0.008), improved disease-specific survival (p = 0.007) and forms an independent prognostic factor for survival (p = 0.033). The intraepithelial CD8+ T-cell and regulatory T-cell (Treg) ratio also forms an independent prognostic factor (p = 0.010) and combination of these two factors reveals a further increased benefit in survival for patients whose tumor displays a dense infiltration with intraepithelial matured M1 macrophages and a high CD8 T-cell/Treg ratio, indicating that both populations of immune cells simultaneously improve survival. Subsequently, we made a heatmap including all known immune parameters for these patients, whereby we were able to identify different immune signatures in CxCa. These results indicate that reinforcement and activation of the intratumoral M1 macrophages may form an attractive immunotherapeutic option in CxCa.

The central distribution of primary afferents from the external eyelids, conjunctiva, and cornea in the rabbit, studied using WGA-HRP and B-HRP as transganglionic tracers.

We have analyzed the afferent limb of the eyeblink and nictitating membrane response of the rabbit by tracing the central distribution of primary afferents from the periorbital skin, conjunctiva, and cornea using horseradish peroxidase agglutinated to wheat germ (WGA-HRP) or conjugated to choleragenoid (B-HRP) as transganglionic tracers. Afferents in the periorbital skin and conjunctiva distribute most heavily to pars caudalis of the spinal trigeminal nucleus (Vc) and to the dorsal horn of spinal segment C1 (dhC1). These afferents terminate predominantly in laminae IIo and IIi and more weakly to the adjacent laminae I and III. There are much weaker projections to spinal segment C2, rostral Vc, and adjacent reticular formation (laminae IV and V) and to the lateral part of pars interpolaris of the spinal trigeminal nucleus (Vi). No conjunctival primary afferents were seen in the rostral divisions of the trigeminal system. Weak afferent inputs from the periorbital skin are present ventrally in pars oralis of the spinal trigeminal nucleus (Vo) and in the principal trigeminal nucleus (Vp). Corneal afferents distribute most densely in the ventral part of Vi and in islands of neuropil within the trigeminal tract at the level of Vi. They also project to caudal Vc and the adjacent dhC1 in laminae I, II, and III. There are sparse projections to the ventral and dorsal parts of Vp and to the ventral part of Vo. Reticular areas adjacent to ventral Vi also receive a few corneal afferents. WGA-HRP- and B-HRP-labeled terminals were distributed similarly in most areas, but lamina I of Vc received terminals labeled with WGA-HRP and Vp and Vo received cutaneous afferents labeled with B-HRP only. Since all subdivisions of the trigeminal system receive periocular and corneal afferent inputs, we suggest that all these subdivisions may be involved in reflex eyeblinks in the rabbit.

Trigeminal inputs to eyeblink motoneurons in the rabbit.

The rabbit nictitating membrane and eyeblink response is widely used in studies of classical conditioning. Eyeblinks involve coordinated activation of the orbicularis oculi motoneurons (OOcVII) and accessory abducens motoneurons (AccVI) which close the external eyelids and nictitating membrane, respectively, and inhibition of levator palpebrae motoneurons (LPIII) whose activity raises the upper eyelid. The identification of blink interneurons that may coordinate these responses is an important step in the analysis of mechanisms supporting eyeblink conditioning as they are likely to receive convergent inputs from circuitry associated with learned as well as unlearned responses. We first investigated the distribution of OOcVII motoneurons in the facial nucleus and LPIII motoneurons in the oculomotor nucleus by retrograde tracing of wheat germ-agglutinated horseradish peroxidase (WGA-HRP) injected into the appropriate muscles. We then used an anterograde tracing method to locate trigeminal and paratrigeminal inputs to OOcVII, to AccVI nucleus, and to LPIII. Injections of WGA-HRP were placed into the principal trigeminal nucleus (Vp) and into all divisions of the spinal trigeminal nucleus. We found an area in Vp and the adjacent rostral parts of pars oralis of the spinal trigeminal nucleus that gave clear projections to OOcVII and AccVI motoneurons and adjacent to LPIII motoneurons in the contralateral oculomotor nucleus. We suggest that neurons in this premotor blink area in rabbits can coordinate learned and reflex blink responses involving the external eyelids and the nictitating membrane. In addition, there are direct projections from the pars interpolaris and pars caudalis of the spinal trigeminal nucleus to the facial nucleus that may mediate short latency responses of the external eyelid orbicularis oculi muscle alone.

Afferent projections to the orbicularis oculi motoneuronal cell group. An autoradiographical tracing study in the cat.

The motoneurons innervating the orbicularis oculi muscle from a subgroup within the facial nucleus, called the intermediate facial subnucleus. This makes it possible to study afferents to these motoneurons by means of autoradiographical tracing techniques. Many different injections were made in the brainstem and diencephalon and the afferent projections to the intermediate facial subnucleus were studied. The results indicated that these afferents were derived from the following brainstem areas: the dorsal red nucleus and the mesencephalic tegmentum dorsal to it; the olivary pretectal nucleus and/or the nucleus of the optic tract; the dorsolateral pontine tegmentum (parabrachial nuclei and nucleus of Kölliker-Fuse) and principal trigeminal nucleus; the ventrolateral pontine tegmentum at the level of the motor trigeminal nucleus; the caudal medullary medial tegmentum; the lateral tegmentum at the level of the rostral pole of the hypoglossal nucleus and the ventral part of the trigeminal nucleus and the nucleus raphe pallidus and caudal raphe magnus including the adjoining medullary tegmentum. These latter projections probably belong to a general motoneuronal control system. The mesencephalic projections are mainly contralateral, the caudal pontine and upper medullary lateral tegmental projections are mainly ipsilateral and the caudal medullary projections are bilateral. It is suggested that the different afferent pathways subserve different functions of the orbicularis oculi motoneurons. Interneurons in the dorsolateral pontine and lateral medullary tegmentum may serve as relay for cortical and limbic influences on the orbicularis oculi musculature, while interneurons in the ventrolateral pontine and caudal medullary tegmentum may take part in the neuronal organization of the blink reflex.

Anatomical observations on the afferent projections to the retractor bulbi motoneuronal cell group and other pathways possibly related to the blink reflex in the cat.

In the cat retractor bulbi (RB) muscle reflexively retracts the eye ball into the orbit. This reflex action is called the nictitating membrane response which, together with the reflex contraction of the orbicularis oculi muscle, constitutes the blink reflex. The retractor bulbi (RB) motoneuronal nucleus is a small cell group located in the lateral tegmentum of the caudal pons, just dorsal to the superior olivary complex. The nucleus is identical to the accessory abducens nucleus and sends its fibers through the abducens nerve. Autoradiographical tracing results indicate that the RB nucleus receives some fibers from the principal and rostral spinal trigeminal nuclei and from the dorsal red nucleus and dorsally adjoining tegmentum. The same areas project to the intermediate facial subnucleus, containing motoneurons innervating the orbicularis oculi muscle. It is suggested that the trigeminal projections take part in the anatomical framework for the R1 component of the blink reflex. Two other brainstem areas i.e.: a portion of the caudal pontine ventrolateral tegmental field and the medullary medial tegmentum at the level of the hypoglossal nucleus were also found to project to the RB motoneuronal cell group and to the intermediate facial subnucleus. These projections were much stronger than those derived from the trigeminal nuclei and red nucleus. Moreover, the medullary premotor area projects not only to the blink motoneuronal cell groups but also to the pontine premotor area. It is suggested that both areas are involved in the R2 blink reflex component. The medullary blink premotor area receives afferents especially from oculomotor control structures in the reticular formation of the brainstem while the pontine blink premotor area receives afferents from the olivary pretectal nucleus and/or the nucleus of the optic tract and from the dorsal red nucleus and its dorsally adjoining area. Because the oculomotor control structures in the reticular formation (by way of the superior colliculus) and the red nucleus receive afferents from trigeminal nuclei, they may play an important role in tactually induced reflex blinking, while the pretectum could take part in the neuronal framework of the visually induced blink reflex.