The impact of hybrid immunity on immune responses after SARS-CoV-2 vaccination in persons with multiple sclerosis treated with disease-modifying therapies.

BACKGROUND AND PURPOSE: Hybrid immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develops from a combination of natural infection and vaccine-generated immunity. Multiple sclerosis (MS) disease-modifying therapies (DMTs) have the potential to impact humoral and cellular immunity induced by SARS-CoV-2 vaccination and infection. The aims were to compare antibody and T-cell responses after SARS-CoV-2 mRNA vaccination in persons with MS (pwMS) treated with different DMTs and to assess differences between naïvely vaccinated pwMS and pwMS with hybrid immunity vaccinated following a previous SARS-CoV-2 infection. METHODS: Antibody and T-cell responses were determined in pwMS at baseline and 4 and 12 weeks after the second dose of SARS-CoV-2 vaccination in 143 pwMS with or without previous SARS-CoV-2 infection and 40 healthy controls (HCs). The MS cohort comprised natalizumab (n = 22), dimethylfumarate (n = 23), fingolimod (n = 38), cladribine (n = 30), alemtuzumab (n = 17) and teriflunomide (n = 13) treated pwMS. Immunoglobulin G antibody responses to SARS-CoV-2 antigens were measured using a multiplex bead assay and FluoroSpot was used to assess T-cell responses (interferon γ and interleukin 13). RESULTS: Humoral and T-cell responses to vaccination were comparable between naïvely vaccinated HCs and pwMS treated with natalizumab, dimethylfumarate, cladribine, alemtuzumab and teriflunomide, but were suppressed in fingolimod-treated pwMS. Both fingolimod-treated pwMS and HCs vaccinated following a previous SARS-CoV-2 infection had higher antibody levels 4 weeks after vaccination compared to naïvely vaccinated individuals. Antibody and interferon γ levels 12 weeks after vaccination were positively correlated with time from last treatment course of cladribine. CONCLUSION: These findings are of relevance for infection risk mitigation and for vaccination strategies amongst pwMS undergoing DMT.

Glia from the central and peripheral nervous system are differentially affected by paclitaxel chemotherapy via modulating their neuroinflammatory and neuroregenerative properties.

Glia are critical players in defining synaptic contacts and maintaining neuronal homeostasis. Both astrocytes as glia of the central nervous system (CNS), as well as satellite glial cells (SGC) as glia of the peripheral nervous system (PNS), intimately interact with microglia, especially under pathological conditions when glia regulate degenerative as well as regenerative processes. The chemotherapeutic agent paclitaxel evokes peripheral neuropathy and cognitive deficits; however, the mechanisms underlying these diverse clinical side effects are unclear. We aimed to elucidate the direct effects of paclitaxel on the function of astrocytes, microglia, and SGCs, and their glia-glia and neuronal-glia interactions. After intravenous application, paclitaxel was present in the dorsal root ganglia of the PNS and the CNS of rodents. In vitro, SGC enhanced the expression of pro-inflammatory factors and reduced the expression of neurotrophic factor NT-3 upon exposure to paclitaxel, resulting in predominantly neurotoxic effects. Likewise, paclitaxel induced a switch towards a pro-inflammatory phenotype in microglia, exerting neurotoxicity. In contrast, astrocytes expressed neuroprotective markers and increasingly expressed S100A10 after paclitaxel exposure. Astrocytes, and to a lesser extent SGCs, had regulatory effects on microglia independent of paclitaxel exposure. Data suggest that paclitaxel differentially modulates glia cells regarding their (neuro-) inflammatory and (neuro-) regenerative properties and also affects their interaction. By elucidating those processes, our data contribute to the understanding of the mechanistic pathways of paclitaxel-induced side effects in CNS and PNS.

Persistent hypokalaemia and intermittent muscle weakness.

A man in his 20s gave a 9-year history of recurrent muscle pain and weakness, occurring mostly after exercise, and lasting for up to 2 days. There had been one episode of severe rhabdomyolysis after cold exposure. He also had longstanding hypokalaemia, which was key to his correct diagnosis but was not followed. This case highlights the importance of an appropriately methodical investigation of weak hypokalaemic patients, and the relevance of hypokalaemia as a cause of neuromuscular symptoms not related to muscular channelopathies.

Osteopontin regulates proliferation, migration, and survival of astrocytes depending on their activation phenotype.

The glycoprotein osteopontin is highly upregulated in central nervous system (CNS) disorders such as ischemic stroke. Osteopontin regulates cell growth, cell adhesion, homeostasis, migration, and survival of various cell types. Accordingly, osteopontin is considered an essential regulator of regeneration and repair in the ischemic milieu. Astrocytes are the most abundant cells in the CNS and play significant roles in health and disease. Astrocytes are involved in homeostasis, promote neuroprotection, and regulate synaptic plasticity. Upon activation, astrocytes may adopt different phenotypes, termed A1 and A2. The direct effects of osteopontin on astrocytes, especially in distinct activation states, are yet unknown. The current study aimed to elucidate the impact of osteopontin on resting and active astrocytes. We established an inflammatory in vitro model of activated (A1) primary astrocytes derived from neonatal wistar rats by exposure to a distinct combination of proinflammatory cytokines. To model ischemic stroke in vitro, astrocytes were subjected to oxygen and glucose deprivation (OGD) in the presence or absence of osteopontin. Osteopontin modulated the activation phenotype by attenuating A1- and restoring A2-marker expression without compromising the active astrocytes' immunocompetence. Osteopontin promoted the proliferation of active and the migration of resting astrocytes. Following transient OGD, osteopontin mitigated the delayed ongoing death of primary astrocytes, promoting their survival. Data suggest that osteopontin differentially regulates essential functions of resting and active astrocytes and confirm a significant regulatory role of osteopontin in an in vitro ischemia model. Furthermore, the data suggest that osteopontin constitutes a promising target for experimental therapies modulating neuroregeneration and repair.

Translating Functional Connectivity After Stroke: Functional Magnetic Resonance Imaging Detects Comparable Network Changes in Mice and Humans.

Background and Purpose: The translational roadblock has long impeded the implementation of experimental therapeutic approaches for stroke into clinical routine. Considerable interspecies differences, for example, in brain anatomy and function, render comparisons between rodents and humans tricky, especially concerning brain reorganization and recovery of function. We tested whether stroke-evoked changes in neural networks follow similar patterns in mice and patients using a systems-level perspective. Methods: We acquired resting-state functional magnetic resonance imaging data during the early poststroke phase in a sample of human patients and compared the observed network changes with data from 2 mouse stroke models, that is, photothrombosis and distal middle cerebral artery occlusion. Importantly, data were subjected to the same processing steps, allowing a direct comparison of global network changes using graph theory. Results: We found that network parameters computed for both mouse models of stroke and humans follow a similar pattern in the postacute stroke phase. Parameters indicating the global communication structure's facilitation, such as small worldness and characteristic path length, were similarly changed in humans and mice in the first days after stroke. Additionally, small worldness correlated with concurrent motor impairment in humans. Longitudinal observation in the subacute phase revealed a negative correlation between initial small worldness and motor recovery in mice. Conclusions: We show that network measures based on resting-state functional magnetic resonance imaging data after stroke obtained in mice and humans share notable features. The observed network alterations could serve as therapeutic readout parameters for future translational studies in stroke research.

Substrate Elasticity Exerts Functional Effects on Primary Microglia.

Microglia-the brain's primary immune cells-exert a tightly regulated cascade of pro- and anti-inflammatory effects upon brain pathology, either promoting regeneration or neurodegeneration. Therefore, harnessing microglia emerges as a potential therapeutic concept in neurological research. Recent studies suggest that-besides being affected by chemokines and cytokines-various cell entities in the brain relevantly respond to the mechanical properties of their microenvironment. For example, we lately reported considerable effects of elasticity on neural stem cells, regarding quiescence and differentiation potential. However, the effects of elasticity on microglia remain to be explored.Under the hypothesis that the elasticity of the microenvironment affects key characteristics and functions of microglia, we established an in vitro model of primary rat microglia grown in a polydimethylsiloxane (PDMS) elastomer-based cell culture system. This way, we simulated the brain's physiological elasticity range and compared it to supraphysiological stiffer PDMS controls. We assessed functional parameters of microglia under "resting" conditions, as well as when polarized towards a pro-inflammatory phenotype (M1) by lipopolysaccharide (LPS), or an anti-inflammatory phenotype (M2) by interleukin-4 (IL-4). Microglia viability was unimpaired on soft substrates, but we found various significant effects with a more than two-fold increase in microglia proliferation on soft substrate elasticities mimicking the brain (relative to PDMS controls). Furthermore, soft substrates promoted the expression of the activation marker vimentin in microglia. Moreover, the M2-marker CD206 was upregulated in parallel to an increase in the secretion of Insulin-Like Growth Factor-1 (IGF-1). The upregulation of CD206 was abolished by blockage of stretch-dependent chloride channels. Our data suggest that the cultivation of microglia on substrates of brain-like elasticity promotes a basic anti-inflammatory activation state via stretch-dependent chloride channels. The results highlight the significance of the omnipresent but mostly overlooked mechanobiological effects exerted on microglia and contribute to a better understanding of the complex spatial and temporal interactions between microglia, neural stem cells, and glia, in health and disease.

The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function.

BACKGROUND: Microglia are essential to maintain cell homeostasis in the healthy brain and are activated after brain injury. Upon activation, microglia polarize towards different phenotypes. The course of microglia activation is complex and depends on signals in the surrounding milieu. Recently, it has been suggested that microglia respond to ion currents, as a way of regulating their activity and function. METHODS AND RESULTS: Under the hypothesis that HCN and KCNQ/Kv7 channels impact on microglia, we studied primary rat microglia in the presence or absence of specific pharmacological blockade or RNA silencing. Primary microglia expressed the subunits HCN1-4, Kv7.2, Kv7.3, and Kv7.5. The expression of HCN2, as well as Kv7.2 and Kv7.3, varied among different microglia phenotypes. The pharmacological blockade of HCN channels by ZD7288 resulted in cell depolarization with slowly rising intracellular calcium levels, leading to enhanced survival and reduced proliferation rates of resting microglia. Furthermore, ZD7288 treatment, as well as knockdown of HCN2 RNA by small interfering RNA, resulted in an attenuation of later microglia activation-both towards the anti- and pro-inflammatory phenotype. However, HCN channel inhibition enhanced the phagocytic capacity of IL4-stimulated microglia. Blockade of Kv7/KCNQ channel by XE-991 exclusively inhibited the migratory capacity of resting microglia. CONCLUSION: These observations suggest that the HCN current contributes to various microglia functions and impacts on the course of microglia activation, while the Kv7/KCNQ channels affect microglia migration. Characterizing the role of HCN channels in microglial functioning may offer new therapeutic approaches for targeted modulation of neuroinflammation as a hallmark of various neurological disorders.

Crosstalk between stressed brain cells: direct and indirect effects of ischemia and aglycemia on microglia.

BACKGROUND: In cerebral ischemia, microglia have a dichotomous role in keeping the balance between pro- and anti-inflammatory mediators to avoid deleterious chronic inflammation and to leverage repair processes. METHODS: We examined functional and inflammatory markers in primary rat microglia in vitro after oxygen-glucose deprivation (OGD) or glucose deprivation (aglycemia). We then investigated the preconditioning effect of OGD or aglycemia upon a subsequent strong inflammatory stimulus, here lipopolysaccharides (LPS). Moreover, an "in vitro brain model" of neurons and glia, differentiated from primary rat neural stem cells, was exposed to OGD or aglycemia. Conditioned medium (CM) of this neuronal/glial co-culture was then used to condition microglia, followed by LPS as a "second hit." RESULTS: OGD or aglycemia at sublethal doses did not significantly affect microglia function, including the expression of inflammatory markers. However, preconditioning with either OGD or aglycemia led to a decreased pro-inflammatory response to a subsequent stimulus with LPS. Interestingly, the anti-inflammatory markers IGF-1 and IL-10 were additionally reduced after such preconditioning, while expression of CD206 remained unaffected. Treatment with CM from the neuronal/glial co-culture alone did not affect the expression of inflammatory markers in microglia. In contrast, treatment with CM increased the expression of both pro- and anti-inflammatory markers in microglia upon a second hit with LPS. Interestingly, this effect could be attenuated in microglia treated with CM from neuronal/glia co-cultures preconditioned with OGD or aglycemia. CONCLUSIONS: Data suggest specific and distinct microglia signatures in response to metabolic stress. While metabolic stress directly and indirectly applied to microglia did not mitigate their subsequent response to inflammation, preconditioning with metabolic stress factors such as OGD and aglycemia elicited a decreased inflammatory response to a subsequent inflammation stimulus.

Transcranial Current Stimulation Alters the Expression of Immune-Mediating Genes.

Despite its extensive use in clinical studies, the molecular mechanisms underlying the effects of transcranial direct current stimulation (tDCS) remain to be elucidated. We previously described subacute effects of tDCS on immune- and stem cells in the rat brain. To investigate the more immediate effects of tDCS regulating those cellular responses, we treated rats with a single session of either anodal or cathodal tDCS, and analyzed the gene expression by microarray; sham-stimulated rats served as control. Anodal tDCS increased expression of several genes coding for the major histocompatibility complex I (MHC I), while cathodal tDCS increased the expression of the immunoregulatory protein osteopontin (OPN). We confirmed the effects of gene upregulation by immunohistochemistry at the protein level. Thus, our data show a novel mechanism for the actions of tDCS on immune- and inflammatory processes, providing a target for future therapeutic studies.

Effect of naturally occurring α-synuclein-antibodies on toxic α-synuclein-fragments.

Alpha-synuclein (α-Syn) is a soluble protein primarily expressed in presynaptic terminals in the central nervous system (CNS). Aggregates of fibrillated α-Syn are the major component of Lewy bodies (LB), a pathologic hallmark of idiopathic Parkinson's disease (PD). Recently, naturally occurring autoantibodies against human α-Syn (nAbs α-Syn) were detected in the peripheral blood of PD patients and controls. Here, we investigated the inhibitory effects of nAbs α-Syn on distinct α-Syn fragments, as well as inflammatory responses and cytotoxicity evoked by nAbs α-Syn in primary microglia. All α-Syn fragments induced the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) from microglia in primary culture. Cotreatment with nAbs α-Syn alleviated the release of pro-inflammatory cytokines induced by α-Syn fragments α-Syn 1-95, α-Syn 61-140, α-Syn 96-140 and α-Syn 112. Treatment with the α-Syn fragments α-Syn 1-95, α-Syn 61-140 and α-Syn 112 impaired the viability of primary microglia. This effect could not be counteracted by cotreatment with nAbs α-Syn. Data suggest an important role of nAbs α-Syn in the α-Syn-induced inflammation cascade, and indicate the potential importance of nAbs in the pathogenesis of PD. This could provide an experimental therapeutic target for patients with PD.

Substrate elasticity induces quiescence and promotes neurogenesis of primary neural stem cells-A biophysical in vitro model of the physiological cerebral milieu.

In the brain, neural stem cells (NSC) are tightly regulated by external signals and biophysical cues mediated by the local microenvironment or "niche." In particular, the influence of tissue elasticity, known to fundamentally affect the function of various cell types in the body, on NSC remains poorly understood. We, accordingly, aimed to characterize the effects of elastic substrates on critical NSC functions. Primary rat NSC were grown as monolayers on polydimethylsiloxane- (PDMS-) based gels. PDMS-coated cell culture plates, simulating the physiological microenvironment of the living brain, were generated in various degrees of elasticity, ranging from 1 to 50 kPa; additionally, results were compared with regular glass plates as usually used in cell culture work. Survival of NSC on the PDMS-based substrates was unimpaired. The proliferation rate on 1 kPa PDMS decreased by 45% compared with stiffer PMDS substrates of 50 kPa (p < 0.05) whereas expression of cyclin-dependent kinase inhibitor 1B/p27Kip1 increased more than two fold (p < 0.01), suggesting NSC quiescence. NSC differentiation was accelerated on softer substrates and favored the generation of neurons (42% neurons on 1 kPa PDMS vs. 25% on 50 kPa PDMS; p < 0.05). Neurons generated on 1 kPa PDMS showed 29% longer neurites compared with those on stiffer PDMS substrates (p < 0.05), suggesting optimized neuronal maturation and an accelerated generation of neuronal networks. Data show that primary NSC are significantly affected by the mechanical properties of their microenvironment. Culturing NSC on a substrate of brain-like elasticity keeps them in their physiological, quiescent state and increases their neurogenic potential.

The plasticity of primary microglia and their multifaceted effects on endogenous neural stem cells in vitro and in vivo.

BACKGROUND: Microglia-the resident immune cells of the brain-are activated after brain lesions, e.g., cerebral ischemia, and polarize towards a classic "M1" pro-inflammatory or an alternative "M2" anti-inflammatory phenotype following characteristic temporo-spatial patterns, contributing either to secondary tissue damage or to regenerative responses. They closely interact with endogenous neural stem cells (NSCs) residing in distinct niches of the adult brain. The current study aimed at elucidating the dynamics of microglia polarization and their differential effects on NSC function. RESULTS: Primary rat microglia in vitro were polarized towards a M1 phenotype by LPS, or to a M2 phenotype by IL4, while simultaneous exposure to LPS plus IL4 resulted in a hybrid phenotype expressing both M1- and M2-characteristic markers. M2 microglia migrated less but exhibit higher phagocytic activity than M1 microglia. Defined mediators switched microglia from one polarization state to the other, a process more effective when transforming M2 microglia towards M1 than vice versa. Polarized microglia had differential effects on the differentiation potential of NSCs in vitro and in vivo, with M1 microglia promoting astrocytogenesis, while M2 microglia supported neurogenesis. Regardless of their polarization, microglia inhibited NSC proliferation, increased NSC migration, and accelerated NSC differentiation. CONCLUSION: Overall, this study shed light on the complex conditions governing microglia polarization and the effects of differentially polarized microglia on critical functions of NSCs in vitro and in vivo. Refining the understanding of microglia activation and their modulatory effects on NSCs is likely to facilitate the development of innovative therapeutic concepts supporting the innate regenerative capacity of the brain.

Bioluminescence imaging visualizes osteopontin-induced neurogenesis and neuroblast migration in the mouse brain after stroke.

BACKGROUND: Osteopontin (OPN), an acidic phosphoglycoprotein, is upregulated in the brain after cerebral ischemia. We previously reported that OPN supports migration, survival, and proliferation of neural stem cells (NSC) in primary cell culture, as well as their differentiation into neurons. We here analyzed the effects of OPN on neuroblasts in vivo in the context of cerebral ischemia. METHODS: Transgenic mice expressing luciferase under the control of the neuroblast-specific doublecortin (DCX)-promoter, allowing visualization of neuroblasts in vivo using bioluminescence imaging (BLI), were injected with OPN intracerebroventricularly while control mice were injected with vehicle buffer. To assess the effects of OPN after ischemia, additional mice were subjected to photothrombosis and injected with either OPN or vehicle. RESULTS: OPN enhanced the migration of neuroblasts both in the healthy brain and after ischemia, as quantified by BLI in vivo. Moreover, the number of neural progenitors was increased following OPN treatment, with the maximum effect on the second day after OPN injection into the healthy brain, and 14 days after OPN injection following ischemia. After ischemia, OPN quantitatively promoted the endogenous, ischemia-induced neuroblast expansion, and additionally recruited progenitors from the contralateral hemisphere. CONCLUSIONS: Our results strongly suggest that OPN constitutes a promising substance for the targeted activation of neurogenesis in ischemic stroke.

The role of dopaminergic immune cell signalling in poststroke inflammation.

Upon ischaemic stroke, brain-resident and peripheral immune cells accumulate in the central nervous system (CNS). Interestingly, these cells express pattern specific to neurotransmitter receptors and, therefore, seem to be susceptible to neurotransmitter stimulation, potentially modulating their properties and functions. One of the principal neurotransmitters in the CNS, dopamine, is involved in the regulation of processes of brain development, motor control and higher brain functions. It is constantly released in the brain and there is experimental and clinical evidence that dopaminergic signalling is involved in recovery of lost neurological function after stroke. Independent studies have revealed specific but different patterns of dopamine receptor subtypes on different populations of immune cells. Those patterns are dependent on the activation status of cells. Generally, exposure to dopamine or dopamine receptor agonists decreases detrimental actions of immune cells. In contrast, a reduction of dopaminergic inputs perpetuates a pro-inflammatory state associated with increased release of pro-inflammatory molecules. In addition, subsets of immune cells have been identified to synthesize and release dopamine, suggesting autoregulatory mechanisms. Evidence supports that inflammatory processes activated following ischaemic stroke are modulated by dopaminergic signalling.

Brain Glucose Metabolism in Parry-Romberg Syndrome.

Parry-Romberg syndrome is a rare disorder characterized by a progressive facial hemiatrophy of the skin, subcutaneous tissue, musculature, bone, and cartilage. It is often associated with neurological symptoms such as trigeminal neuropathy, paresthesia of the face, migraine, and seizures and can be paired with ocular problems and ipsilateral progressive body atrophy. Here, we present a young woman with progressive facial hemiatrophy, who was referred for FDG-PET/CT. Hypometabolism was observed in the left cingulate and postcentral gyrus, left cerebellum, and right basal ganglia. Hypometabolism may be observed before anatomical changes and therefore facilitate early diagnosis.

Osteopontin directly modulates cytokine expression of primary microglia and increases their survival.

Osteopontin (OPN) is constitutively expressed in the brain and upregulated during neuroinflammation, e.g., focal cerebral ischemia. In OPN-deficient mice, microglia are deregulated after ischemia, but specific OPN-effects on microglia remain elusive. Primary microglia were cultured in the presence or absence of OPN. The survival of microglia under stress conditions was dose-dependently increased by OPN. Lipopolysaccharides (LPS)-induced release of nitric oxide (NO), TNF-α, and IL-6, as well as expression of inducible Nitric Oxide Synthase (iNOS), were attenuated by OPN. Data suggest that OPN modulates microglia function by shifting their inflammatory profile towards a neutral anti-inflammatory phenotype.

Osteopontin mediates survival, proliferation and migration of neural stem cells through the chemokine receptor CXCR4.

INTRODUCTION: Osteopontin (OPN) is a phosphoglycoprotein with important roles in tissue homeostasis, wound healing, immune regulation, and stress responses. It is expressed constitutively in the brain and upregulated during neuroinflammatory responses; for example, after focal cerebral ischemia. To date, its effects on neural stem cells (NSC) remain to be elucidated and are, accordingly, the subject of this study. METHOD: Primary fetal rat NSC were cultured as homogenous monolayers and treated with different concentrations of OPN. Fundamental properties of NSC were assessed following OPN exposure, including proliferative activity, survival under oxidative stress, migration, and differentiation potential. To elucidate a putative action of OPN via the CXC chemokine receptor type 4 (CXCR4), the latter was blocked with AMD3100. To investigate effects of OPN on endogenous NSC in vivo, recombinant OPN was injected into the brain of healthy adult rats as well as rats subjected to focal cerebral ischemia. Effects of OPN on NSC proliferation and neurogenesis in the subventricular zone were studied immunohistochemically. RESULTS: OPN dose-dependently increased the number of NSC in vitro. As hypothesized, this effect was mediated through CXCR4. The increase in NSC number was due to both enhanced cell proliferation and increased survival, and was confirmed in vivo. Additionally, OPN dose-dependently stimulated the migration of NSC via CXCR4. Moreover, in the presence of OPN, differentiation of NSC led to a significant increase in neurogenesis both in vitro as well as in vivo after cerebral ischemia. CONCLUSION: Data show positive effects of OPN on survival, proliferation, migration, and neuronal differentiation of NSC. At least in part these effects were mediated via CXCR4. Results suggest that OPN is a promising substance for the targeted activation of NSC in future experimental therapies for neurological disorders such as stroke.

Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing.

Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way.