Search

BACKGROUND Lewis serologic reagents frequently give inaccurate phenotyping results. Furthermore these serologic reagents are often used in nonserologic assays such as inhibition and immunohistochemistry. In both scenarios knowledge of the fine specificity and cross-reactivity of these reagents will improve the quality of results obtained. STUDY DESIGN AND METHODS A range of contemporary and historical workshop and developmental Lewis reagents including mouse monoclonal (MoAb) and human and goat polyclonal (PoAb) reagents were evaluated. All were evaluated both against Lewis kodecytes expressing only single Le(a) , Le(b) , ALe(b) , BLe(b) , Le(x) , Le(y) , ALe(y) , or BLe(y) antigens and against the same antigens inkjet printed on a paper-based microplate and analyzed by enzyme immunoassay. Nine clinical samples were also evaluated. A kodecyte antigen dilution sensitivity assay was used to establish the ratio of Le(b) antigen between group A1 /A2 and O RBCs. RESULTS A continuum of cross-reactivity from Le(x) through to H was observed with MoAbs. All PoAb and few MoAb anti-Le(a) samples and reagents cross-reacted to some degree with Le(b) antigen. Some PoAb and MoAb anti-Le(b) did not cross-react with Le(a) . All polyclonal goat anti-Le(b) reagents showed substantial activity against ALe(b) and BLe(b) , while no MoAb reagent had this activity. A1 RBCs had less than half the Le(b) antigen of A2 /O RBCs. CONCLUSIONS Substantial cross-reactivity of both MoAbs and PoAbs with related antigens highlights the risks of using serologic reagents in nonserologic assays or against synthetic antigens. The lack of ALe(b) activity in anti-Le(b) MoAbs explains their poor performance against blood group A1 Le(a-b+) phenotypes.

Background Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. Study Design and Methods Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Lea trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. Results Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. Conclusions All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems.

Inbreeding depression is the reduction of performance caused by mating of close relatives. In livestock populations, inbreeding depression has been traditionally estimated by regression of phenotypes on pedigree inbreeding coefficients. This estimation can be improved by utilising genomic inbreeding coefficients. Here we estimate inbreeding depression for the insect bite hypersensitivity (IBH) prevalence, the most common allergic horse disease worldwide, in Old Kladruber horse. In a deep pedigree with 3,214 horses (187 genotyped) we used a generalized linear mixed model with IBH phenotype from 558 horses examined between 1996 and 2009 (1,368 records). In addition to the classical pedigree information, we used the single-step approach that enabled joint use of pedigree and genomic information to estimate inbreeding depression overall genome and ELA class II (equine leuckocyte antigens) region. Significant inbreeding depression was observed in all models fitting overall inbreeding coefficients (odds ratio between 1.018 and 1.074, P

We have been able to demonstrate that glutaraldehyde fixation of tumor cells or tumor cell membrane fragments protect syngeneic mice against the subsequent challenge with viable cells. To date we have not been able to detect any antibody against the tumor cells in animals so immunized. The specificity of the response is not complete, although a major component of the protective effect of glutaraldehyde-treated tumor cells is immunologically specific.

Normal human diploid cells, such as WE-38, transformed by an oncogenic virus, SV40, are greatly depleted of a cathodal esterase isozyme using both immunological and enzymatic criteria. This cathodal esterase is primate-specific and associated with lysosomes of many different tissue cells. It provides a clear example of antigenic loss associated with malignant change.

Friend leukemia virus (FLV) leukemogenesis was prevented by treatment of the virus with Concanavalin A (Con A). Mice infected with the lectin-treated virus, however, showed evidence of a dormant infection since infectious virus could be recovered for as long as 100 days. Humoral immune responses to sheep erythrocytes (SRBC), a thymus-dependent antigen, and to E. coli lipopolysaccharide (LPS), a thymus-independent antigen, were depressed (approximately 80-90%) in mice given the Con A-treated FLV. Cell transfer studies indicated that the impaired responsiveness to SRBC was related to a defect in B-lymphocyte function, similar to the impairment in mice infected with untreated FLV. The mitogenic response of splenocytes from Con A-FLV mice to E. coli LPS was also depressed as was the ability of Ig-bearing spleen cells to redistribute these immunoglobulin receptors into polar caps. The impaired immune responsiveness in the Con A-FLV infected mice appeared associated with the persistent virus infection and not to neoplastic transformation generally associated with leukemogenic process.

TA3-HA nonspecific tumors growing in ascites form shed their membrane components into the ascites fluid. This product can induce tumor growth enhancement if given together with or after i.p. tumor inoculation in relatively large quantities, but if given i.m. in small quantities, several days before tumor challenge, the mice show an elevated resistance to the tumor. The strictly strain-specific TA3-St line does not release such membrane components into the ascites fluid. It has also been found that the TA3-HA cells have a larger membrane fluidity than the TA3-St cells, as measured by membrane microviscosity, using the Shinitzki procedure. Isolation and gross chemical analysis of the TA3-Ha ascites fluid component was carried out. Results, so far, indicate that the active site of this product is probably carbohydrate in nature. The possible mode of action of such component in tumor growth enhancement is most probably by overwhelming and neutralizing immunocytes capable of recognition and eventually cytotoxic action.

The immune system is subject to an array of identified autoregulatory processes, but immunoregulation may also have a further basis in a network of immune-neuroendocrine interactions. Two antigens each produced an increase of more than 100% in electrical activity of individual neurones in the ventromedial but not in the anterior nucleus of the rat hypothalamus. Animals that failed to respond to antigen manifested no increase in the firing rate. These findings constitute the first evidence for a flow of information from the activated immune system to the hypothalamus, suggesting that the brain is involved in the immune response.

Supramolecular peptide hydrogel has shown promising potential in vaccine development largely because of its ability to function both as antigen depot and immune adjuvant. Nap‐GdFdFdY, a tetrapeptide hydrogel that has been previously reported to exhibit adjuvant effect, is inadvertently found to contain conserved peptide sequence for insulin, proinsulin, and glutamic acid decarboxylase, 3 major autoantigens for the autoimmune type 1 diabetes (T1D). At present, despite being managed clinically with insulin replacement therapy, T1D remains a major health threat with rapidly increasing incidences, especially in children and young adults, and antigen‐specific immune tolerance induction has been proposed as a feasible approach to prevent or delay T1D progression at an early stage. Here, it is reported that innoculation of Nap‐GdFdFdY leads to complete protection of nonobese diabetic (NOD) mice from T1D development till the age of 36 weeks. Better maintenance of pancreatic islet morphology with minimal immune cell infiltration is also observed from mice exposed to Nap‐GdFdFdY. This beneficial impact is mainly due to its facilitative role on enhancing peripheral T regulatory cell (Treg) population, shown as increased splenic Treg percentage, and function, demonstrated by maintenance of circulating TGF‐β1 level. Serum cytokine microarray data further implicate a “buffering” role of Nap‐GdFdFdY on systemic inflammatory tone in NOD mice. Thus, with its versatility, applicability, and excellent potency, Nap‐GdFdFdY is posited as a novel therapeutic intervention for T1D., A hydrogel of Nap‐GdFdFdY containing conserved sequence of autoantigens including insulin, proinsulin, and glutamic acid decarboxylase as a novel therapeutic intervention for the autoimmune type 1 diabetes. Better pancreatic islet morphology with minimal immune cell infiltration is observed from mice with hydrogel because of enhancing peripheral T regulatory cell population and maintenance of circulating TGF‐β1 level.

Nanoparticles (NPs) can serve as a promising vaccine delivery platform for improving pharmacological property and codelivery of antigens and adjuvants. However, NP‐based vaccines are generally associated with complex synthesis and postmodification procedures, which pose technical and manufacturing challenges for tailor‐made vaccine production. Here, modularly programmed, polyethyleneimine (PEI)‐based NP vaccines are reported for simple production of personalized cancer vaccines. Briefly, PEI is conjugated with neoantigens by facile coupling chemistry, followed by electrostatic assembly with CpG adjuvants, leading to the self‐assembly of nontoxic, sub‐50 nm PEI NPs. Importantly, PEI NPs promote activation and antigen cross‐presentation of antigen‐presenting cells and cross‐priming of neoantigen‐specific CD8+ T cells. Surprisingly, after only a single intratumoral injection, PEI NPs with optimal PEGylation elicit as high as ≈30% neoantigen‐specific CD8+ T cell response in the systemic circulation and sustain elevated CD8+ T cell response over 3 weeks. PEI‐based nanovaccines exert potent antitumor efficacy against pre‐established local tumors as well as highly aggressive metastatic tumors. PEI engineering for modular incorporation of neoantigens and adjuvants offers a promising strategy for rapid and facile production of personalized cancer vaccines., Modularly programmed, polyethyleneimine (PEI)‐based nanoparticle (NP) vaccines are developed for simple production of personalized cancer vaccines. PEI‐antigen conjugates and CpG adjuvants are mixed form nontoxic, sub‐50 nm PEI NPs. PEI NPs elicit strong, long‐lasting neoantigen‐specific CD8+ T cell response with potent antitumor efficacy against local as well as metastatic tumors. PEI NPs offer a promising strategy for personalized cancer vaccination.

Using T‐cell chimeric antigen receptors (CAR‐T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor‐associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106‐member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR‐T's produce cytokines known to be associated with T cell activation in a CD38 expression‐dependent manner. This avidity‐based selection endows the engineered T cells with minimal off‐tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR‐T therapy to TAAs previously considered undruggable., A novel polyclonal combinatorial cellular library of chimeric antigen receptors provides a comprehensive coverage of immune responses to specific antigens in vitro. An efficient and effective avidity‐based selection strategy takes advantage of paracrine signaling systems in cell–cell interactions, which endow the engineered T cells with robust antitumor efficacy both in vitro and in vivo with minimal on‐target off‐tumor effects.

Aims To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites. Methods and results EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL‐4, IL‐5 and IL‐13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL‐5 and IL‐9 (exsheated L3 and L4) and IFN‐γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL‐4, IL‐5 and IL‐10 while concomitantly enhancing the expression of IFN‐γ. Conclusion The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.

The immunology of falciparum malaria, the lethal type of human malaria, has been transformed by two developments. First, a culture system for the asexual blood stages of Plasmodium falciparum.1 Secondly, the cloning and expression of genes coding for a large number of the protein antigens of this malaria parasite over the past two years. Data on proteins, protein antigens and epitopes of P. falciparum supplied by gene cloning techniques have been supplemented by monoclonal antibody approaches, peptide synthesis, and high-resolution immunochemistry.

Combined phototherapy and immunotherapy demonstrates strong potential in the treatment of metastatic cancers. An upconversion nanoparticle (UCNP) based antigen‐capturing nanoplatform is designed to synergize phototherapies and immunotherapy. In particular, this nanoplatform is constructed via self‐assembly of DSPE‐PEG‐maleimide and indocyanine green (ICG) onto UCNPs, followed by loading of the photosensitizer rose bengal (RB). ICG significantly enhances the RB‐based photodynamic therapy efficiency of UCNP/ICG/RB‐mal upon activation by a near‐infrared (NIR) laser, simultaneously achieving selective photothermal therapy. Most importantly, tumor‐derived protein antigens, arising from phototherapy‐treated tumor cells, can be captured and retained in situ, due to the functionality of maleimide, which further enhance the tumor antigen uptake and presentation by antigen‐presenting cells. The synergized photothermal, photodynamic, and immunological effects using light‐activated UCNP/ICG/RB‐mal induces a tumor‐specific immune response. In the experiments, intratumoral administration of UCNP/ICG/RB‐mal, followed by noninvasive irradiation with an NIR laser, destroys primary tumors and inhibits untreated distant tumors, using a poorly immunogenic, highly metastatic 4T1 mammary tumor model. With the simultaneous use of anti‐CTLA‐4, about 84% of the treated tumor‐bearing mice achieve long‐term survival and 34% of mice develop tumor‐specific immunity. Overall, this antigen‐capturing nanoplatform provides a promising approach for the treatment of metastatic cancers.

Background Fasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties. Objectives Given the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspecies paratuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co-infection with F. hepatica might have on the course of Johne's disease (JD). Methods This study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products. Results We found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre-stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake. Conclusions Overall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co-stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

Influenza poses a severe threat to global health. Despite the whole inactivated virus (WIV)‐based nasal vaccine being a promising strategy for influenza protection, the mucosal barrier is still a bottleneck of the nasal vaccine. Here, a catalytic mucosal adjuvant strategy for an influenza WIV nasal vaccine based on chitosan (CS) functionalized iron oxide nanozyme (IONzyme) is developed. The results reveal that CS‐IONzyme increases antigen adhesion to nasal mucosa by 30‐fold compared to H1N1 WIV alone. Next, CS‐IONzyme facilitates H1N1 WIV to enhance CCL20‐driven submucosal dendritic cell (DC) recruitment and transepithelial dendrite(TED) formation for viral uptake via the toll‐like receptor(TLR) 2/4‐dependent pathway. Moreover, IONzyme with enhanced peroxidase (POD)‐like activity by CS modification catalyzes a reactive oxygen species (ROS)‐dependent DC maturation, which further enhances the migration of H1N1 WIV‐loaded DCs into the draining lymph nodes for antigen presentation. Finally, CS‐IONzyme‐based nasal vaccine triggers an 8.9‐fold increase of IgA‐mucosal adaptive immunity in mice, which provides a 100% protection against influenza, while only a 30% protection by H1N1 WIV alone. This work provides an antiviral alternative for designing nasal vaccines based on IONzyme to combat influenza infection., A CS‐IONzyme‐based influenza mucosal vaccine with dual targeting of both nasal mucosa and submucosal DCs, leading to a robust immunoprotection against influenza virus is designed. Utilizing an excellent POD‐like activity, a catalytic immune‐adjuvant CS‐IONzyme against the bottleneck of mucosal vaccines is successfully developed by both fully mobilizing submucosal DCs to form TEDs for antigen uptake and strongly activating DC maturation.

Background and Objectives Allogeneic hematopoietic stem cell transplantation (HSCT) is a routine clinical procedure performed to treat patients with haematological malignancies, primary immune deficiencies or metabolic disorders. Infections during lymphopenia after allogeneic HSCT are associated with high mortality and morbidity. Typical infectious agents are Epstein–Barr virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus and fungi. The study aim was to evaluate whether measurement of the responses of antigen-specific T-cells, recognizing infectious pathogens would correlate to protective functions in the stem cell recipient post-transplant. Materials and Methods Twenty-one grafts were analysed by flow cytometry and cells were stimulated in vitro with relevant infectious antigens, followed by evaluation of T-cell proliferation and cytokine production. Results were compared to the recipients’ clinical records 1-year post-transplantation. Results We show that an extensive repertoire of transferred antigen-specific T-cells from allogeneic donor grafts against infectious agents, involved in post-transplant infections, are linked to an absence of infectious complications for the recipient up-to 1-year post-transplant. The protective effect was associated with antigen-specific T-cell proliferation and IL-1β secretion. Conclusion Our results suggest that assaying T-cell function before HSCT could determine individual risks for infectious complications and thus aid in clinical decision-making regarding prophylactic and pre-emptive anti-infective therapy.

Immunotherapy has shown tremendous promise for improving cancer treatment. Unfortunately, antigen‐presenting cells (APCs) in cancer patients cannot effectively recognize and process tumor antigens to activate host immune responses. In this study, an approach is developed to improve cancer immunotherapy that utilizes endogenous antigen‐carrying nanoparticles (EAC‐NPs), which encompasses a set of antigens isolated from solid tumors and adjuvants. The EAC‐NPs specifically target APCs and subsequently result in enhanced T cell responses and improved antitumor efficacy. Mechanistic studies reveal that the EAC‐NPs enhance and prolong the presence of immune compounds in APCs, which ensure persistent antigen loading and stimulation, induce a rapid proliferation of CD4+ and CD8+ T cells, and significantly increase the ratios of intratumoral CD4+ T/Treg and CD8+ T/Treg. The work using nanotechnology provides a promising strategy in improving antitumor immunity by enhancing the immunogenicity and presentation of tumor self‐antigens for cancer immunotherapy., Endogenous antigen‐carrying nanoparticles are phagocytosed by antigen‐presenting cells (APCs) triggering antigen and Toll‐like receptor signaling. Then the APCs induce highly efficient differentiation of T cells toward helper T (CD4+) cells and cytotoxic T lymphocytes (CD8+) to finally generate potent antitumor immune responses. A promising strategy is established for cancer immunotherapy by enhancing the immunogenicity and presentation of tumor self‐antigens.

Due to specific immune recognition receptors on the surface of T cells, their membranes are promising mimic nanocarriers for delivering drugs to tumor lesions. However, this single targeting strategy potentially compromises therapy efficacy for tumor targeting due to inter‐ and intra‐heterogeneity of tumors. Azide (N3) or bicyclo [6.1.0] nonyne (BCN) modified unnatural sugars can be successfully incorporated into surface glycans of various tumor cells as artificial receptors, which is expected to overcome the insufficiency of single targeting. Based on this artificial tumor targeting strategy, indocyanine green nanoparticles (INPs) coated with N3‐labeled T cell membrane (N3‐TINPs) are constructed, which can specifically target the natural antigen and BCN artificial receptors on tumors through immune recognition and bioorthogonal chemistry, respectively. The results show that the fluorescence intensity in the tumors of mice treated with N3‐TINPs is 1.5 fold compared with that of the mice treated with unlabeled TINPs. The accumulated N3‐TINPs in the tumor significantly increase the photothermal therapeutic effect without adverse effect. Therefore, this T cell membrane mimicking nanoparticles based bioorthogonal chemistry may provide an alternative artificial targeting strategy for further tumor targeting photothermal therapy.

The continued threat of emerging, highly lethal infectious pathogens such as Middle East respiratory syndrome coronavirus (MERS‐CoV) calls for the development of novel vaccine technology that offers safe and effective prophylactic measures. Here, a novel nanoparticle vaccine is developed to deliver subunit viral antigens and STING agonists in a virus‐like fashion. STING agonists are first encapsulated into capsid‐like hollow polymeric nanoparticles, which show multiple favorable attributes, including a pH‐responsive release profile, prominent local immune activation, and reduced systemic reactogenicity. Upon subsequent antigen conjugation, the nanoparticles carry morphological semblance to native virions and facilitate codelivery of antigens and STING agonists to draining lymph nodes and immune cells for immune potentiation. Nanoparticle vaccine effectiveness is supported by the elicitation of potent neutralization antibody and antigen‐specific T cell responses in mice immunized with a MERS‐CoV nanoparticle vaccine candidate. Using a MERS‐CoV‐permissive transgenic mouse model, it is shown that mice immunized with this nanoparticle‐based MERS‐CoV vaccine are protected against a lethal challenge of MERS‐CoV without triggering undesirable eosinophilic immunopathology. Together, the biocompatible hollow nanoparticle described herein provides an excellent strategy for delivering both subunit vaccine candidates and novel adjuvants, enabling accelerated development of effective and safe vaccines against emerging viral pathogens., To improve vaccination efforts against Middle East respiratory syndrome coronavirus (MERS‐CoV), a virus‐mimicking vaccine is herein prepared with a capsid‐like hollow polymeric nanoparticle loaded with STING agonists and coated in MERS‐CoV antigens. The viromimetic nanoparticle facilitates safe and effective vaccination against the lethal virus and offers a versatile platform for combatting emerging infectious threats.

Vaccine coverage is severely limited in developing countries due to inefficient protection of vaccine functionality as well as lack of patient compliance to receive the additional booster doses. Thus, there is an urgent need to design a thermostable vaccine delivery platform that also enables release of the bolus after predetermined time. Here, the formation of injectable and light-activatable polybubbles for vaccine delivery is reported. In vitro studies show that polybubbles enable delayed burst release, irrespective of cargo types, namely small molecule and antigen. The extracorporeal activation of polybubbles is achieved by incorporating near-infrared (NIR)-sensitive gold nanorods (AuNRs). Interestingly, light-activatable polybubbles can be used for on-demand burst release of cargo. In vitro, ex vivo, and in vivo studies demonstrate successful activation of AuNR-loaded polybubbles. Overall, the light-activatable polybubble technology can be used for on-demand delivery of various therapeutics including small molecule drugs, immunologically relevant protein, peptide antigens, and nucleic acids.

Serum γ-globulin levels have been determined by paper electrophoresis in 54 patients with cirrhosis of the liver before and after establishment of a portocaval shunt. As a control group 23 patients operated on for cancer of the lung were studied in the same way. A significant average increase from 18.6 to 23.0 g/1 in the cirrhotics was found, whereas no increase in the control group was observed. Serum immunoglobulins and antibodies to E. coli 0 group antigens were studied in 31 patients with cirrhosis and a portocaval shunt and 128 patients with cirrhosis without portocaval shunt. Significantly higher levels of IgG and E. coli antibodies were found in the patients with a portocaval shunt. It is proposed that these observations can be explained by the change in liver circulation brought about by the operation. If the cirrhotic liver has retained some of the ability of the normal liver to inhibit immunogens as previously proposed by us, the observed changes in immunoglobulins and E. coli antibodies should be expected.

The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367–16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, –0.218 to 0.465). 3–93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. Copyright © 2013 Pathological Society of Great Britain and Ireland.

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O-antigen to different amino acids of CRM197 protein carrier. In particular, two novel methods for site-selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH-controlled transglutaminase-catalyzed modification of lysine, respectively. Importantly, conjugation at the C186-201 bond resulted in significantly higher anti O-antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.

Tumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects. This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR. We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells. We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity. Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641. We further demonstrate that SA1641 did not influence the plasma membrane permeability, indicating an intracellular interaction of SA1641 and the toxin components of targeted toxins. Surface plasmon resonance measurements point to a transient binding of SA1641 to the toxin components of targeted toxins.

Sera from patients with myasthenia gravis contain antibodies to a citric acid extractable antigen present in heart and skeletal muscle. This antigen is located in the sarcolemmal/subsarcolemmal region of the muscle fibres. The antigen is resistant to pH changes between 2 and 10, but is inactivated by treatment at 56° C for 60 min or at 70° C for 30 min. The antigenic determinant is of protein nature.

The idiotypic network can be experimentally altered to induce protective immune responses against microbial pathogens. Both internal image and noninternal image anti‐idiotypic (anti‐Id) antibodies have been shown to trigger antigen (Ag)‐specific immune responses. Therefore, mechanisms of anti‐Id vaccination appear to go beyond structural mimicry of Ag, but remain undefined. Using the neurotropic murine coronavirus animal model, we have previously shown that a polyclonal noninternal image anti‐Id (Ab2) could vaccinate BALB/c mice. To characterize its mode of action, we have examined the immune modulating capability of this Ab2 in vivo in strains of mice with different H‐2 haplotypes. Even though only internal image anti‐Id are expected to induce non‐genetically restricted immunity, this noninternal image Ab2 induced protective immunity in four of eight genetically different strains of mice susceptible to coronavirus infection. These were BALB/c (H‐2d), DBA/1 (H‐2d), DBA/1 (H‐2q), and SWR (H‐2q) mice. Protection was generally correlated with the induction of specific antiviral Ab (Ab3) that showed biological properties, such as virus neutralization in vitro, similar to the initial Ab1. To evaluate the genetic implication of the H‐2 haplotypes in protection, congenic mice were also tested. Vaccination profiles suggest that cooperation between background gene(s) of the BALB/c mouse with H‐2d and H‐2q loci is necessary for an optimal protective immune response, although the main genetic element(s) regulating the antiviral response to Ab2 inoculation appeared to be located outside the major histocompatibility complex. These results are consistent with the ability of Ab2 to induce protective antiviral antibodies in genetically different animals by biological mimicry.

Summary The initial lung lesions in two calves intrabronchially inoculated with Haemophilus somnus are described. The animals were euthanized within 7 h after challenge. The in situ location of H. somnus and accompanying lesions were examined by light microscopy, immunohistochemistry and transmission electron microscopy (TEM). Inoculation with H. somnus resulted in the development of acute pulmonary lesions within 3.5 h. H. somnus antigen was demonstrated only within the luminal spaces of the airways and in one area of bronchio‐associated lymphoid tissue (BALT). As observed by TEM, the bacteria were phagocytized by both neutrophils and alveolar macrophages. Antigen was never demonstrated in the pulmonary intravascular macrophages.

We have examined the expression on a panel of 22 human kidneys of polymorphic B-cell determinants recognized by mouse monoclonal antibodies. Monoclonal antibodies from a mouse immunized with an antigenic preparation from a DR4 positive B-cell line reacted preferentially with kidneys from DR4 positive donors (p less than 0.005), and the pattern of reactivity with kidney tissues was similar to that of antibodies to monomorphic determinants of class II. However, these antibodies did not show clear specificity for DR4 on lymphocytes in standard serological analyses. These results provide evidence for the expression of polymorphic class II determinants on human kidneys. Reasons for the differences in the apparent specificities of the monoclonal antibodies when tested on kidney sections and lymphocytes are discussed.

Three previously defined antigenic subsets of C3 (C3(S), C3(D) alpha and C3(D)beta) clearly distinguish native C3 from C3 that has been denatured in the presence of sodium dodecyl sulphate (C3-SDS): C3-SDS expresses antigens of all 3 subsets, while native C3 only expressed C3(S) antigens. The radioimmunometric assessment performed in this study revealed that 90% of the C3(S) and C3(D)alpha and 70% of the C3(D)beta antigens demonstrable in C3-SDS also was expressed by physiologically bound C3b on sheep erythrocytes. The antigenic properties of bound C3b were not shared by soluble C3b, which (like soluble C3) only expressed C3(S) antigens, nor did soluble C3b 'spontaneously' released from complement-treated target cells express antigens other than C3(S). We therefore conclude that the expression of C3(D) antigens, under physiological conditions of activation, reflects a conformational modification unique for bound C3b and occurring to an extent comparable to that of C3 in the presence of a strong denaturant. Additional studies further revealed that the antigenic profile of bound C3b is determined by a heterogeneous population of molecules differing in their relative expression of the C3(S), C3(D)alpha and C3(D)beta subsets.

The antigenicity of human syncytiotrophoblast membrane (TM) was analyzed using rabbit antiserum raised against TM. From immunoblot analysis, about ten protein bands in TM were recognized by the anti-TM. These included placental alkaline phosphatase and TM-bound albumin. From limited antigenic specificity studies, these antigens, with the exception of albumin, were not detectable in membranes of liver, kidney, heart, and erythrocyte, or in human normal serum. Therefore, these antigens appear to be placental membrane specific. Analysis of lymphocyte membrane by Immunoelectrophoresis, crossed Immunoelectrophoresis, and immunoblot techniques revealed that a single protein band, designated “40 kDa,” was cross-reactive with the anti-TM antiserum, indicating that this antigen is shared commonly between placenta and lymphocyte membranes. Experimental evidence suggesting that the 40-kDa membrane antigen is probably a unique trophoblast-Iymphocyte cross-reactive antigen is based on the following observations: 1) the 40-kDa antigen was not detectable in liver, heart, and kidney membrane; 2) γ2-microglobulin (12,000 daltons) was not detectable in our TM preparation; and 3) the lymphocyte membrane showed only a single protein band at 40,000 daltons, not at 12,000 for γ-microglobulin.

In order to determine the structure of the tumor-specific immunodominant group of the carcinoembryonic antigen (CEA) of human gastro-intestinal tumors, immunologically active fragments from this glycoprotein were prepared by partial enzymatic hydrolysis using the proteolytic enzyme nagase. The fragments were purified by sequential molecular sieve, adsorption and ion exchange chromatography, and were analysed for carbohydrate and amino-acid composition by gas-liquid chromatography. The antigenic activity of each fragment was tested using both the Z-gel and Farr radioimmunoassay techniques for CEA. Immunologically active glycopeptides with molecular weights of 1,000–5,000 Daltons, which contained the tumor antigenic determinant of CEA, had relatively high contents of N-acetyl-D-glucosamine, aspartic acid or asparagine, and glutamic acid or glutamine.

A purification procedure applied to H-2 antigens (controlled by the H-2 locus) was used for the characterization of HL-A substances (HL-A2 and HL-A7) (controlled by the HL-A locus) solubilized from lymphoid cell membranes by delipidation. Upon Sephadex chromatography the serologically active material had an apparent Mr of 32000, and displayed, in relation to H-2 antigens, a faster electrophoretic mobility and higher pI values. One sulfhydryl group and one (intrachain) disulfide bond were present in partially purified HL-A preparations. Reduction and aminoethylation in 4 M urea did not influence the electrophoretic migration of HL-A active substances, suggesting that, as already demonstrated for H-2 alloantigens, in HL-A molecules the genetic determinants are present on single polypeptide chains. Highly purified HL-A alloantigens, like H-2 substances, revealed a high degree of microheterogeneity. These findings further support the concept of the genetic and structural homology of the main histocompatibility systems in mouse and man.

Serum from a patient with multiple myeloma exhibited an unusual concurrence of abnormalities: (1) bitypic gammopathy consisting of a whole Λ-IgG 1 paraprotein plus a circulating Λ-Bence Jones protein; (2) marked insolubility of the partially purified paraprotein under conditions of low ionic strength, but good solubility upon further purification; (3) release of an unusual low molecular weight fragment containing Fc antigenic determinants under standard conditions of mercaptoethanol reduction of the purified paraprotein. Evidence is presented supporting the origin of the Fc fragment from the paraprotein heavy chain and describing the requirement for 6 M urea for its dissociation from the reduced and alkylated heavy chain. The phenomenon is evidently not rare, since screening of 14 additional myeloma sera revealed one with similar euglobulin properties and an apparently similar Fc fragment abnormality. The nature and origin of the fragment in relation to previously described immunoglobulin fragments are discussed.

In order to examine how rat erythrocytes stimulate erythrocyte autoantibody production at the molecular level, we have identified rat erythrocyte antigens by immunoprecipitation and western blotting using monoclonal antibodies and antisera. A novel non-radioactive immunoprecipitation technique was used, which employed biotin as a label and a luminescent detection system. The new method was validated by comparison with conventional immunoprecipitation using 125I. Glycophorins of relative molecular mass (Mr) 81,000 and 38,000 were found to be the major antigenic components of rat erythrocytes, while band 3 (the most abundant erythrocyte membrane protein) was not recognized by rat-specific antibodies. The same surface antigens were recognized by sera from mice producing erythrocyte autoantibodies and by sera from mice in which autoantibody production was suppressed. Nine other minor rat-specific antigens were identified by blotting, ranging in Mr from 23,000 to 147,000. Analysis of the integral membrane proteins of rat and mouse erythrocytes by sodium dodecyl sulphate (SDS) electrophoresis followed by silver or periodic acid-Schiff (PAS) stains revealed differences between the glycophorins, but not between rat and mouse band 3. Thus, the major antigenic differences correspond to discernible biochemical differences between rat and mouse erythrocyte sialoglycoproteins.

The lymphoid cell composition of milky spots was analyzed in unprimed mice before and after i.p. immunization with sheep red blood cells. Milky spots contained surface immunoglobulin-positive B lymphocytes, and T cells of the helper and cytotoxic phenotype. After secondary antigen challenge (a) the number of lymphocytes increased up to 40-fold, (b) B and T cells were found to segregate into distinct areas in situ, and (c) lymphocytes were found to associate with I-A-negative stromal cells in vivo. These findings qualify milky spots as a peripheral lymphoid organ exhibiting a remarkable change in number and composition of lymphocytes in response to a local antigen stimulus.

ALG was produced in rabbits by intracutaneous injections of small doses (106 cells) of C3H mouse thymocytes suspended in Freund's incomplete adjuvant. The immunosuppressive potency of this ‘low-dose’ ALG evaluated by allo-skin graft survival proved to be comparable to that of ALG produced by intravenous injection of high doses of lymphoid cells (ad modum Levey & Medawar). In addition the ‘low-dose’ method gave rise to negligible amounts of haemagglutinins, lymphagglutinins, and lymphocytotoxic antibodies. The ‘low-dose’ method may represent a major advantage in overcoming the limiting factor presented by scarcity of available antigen, which is regularly the case in human ALG production.

Structural analysis of a representative group of Shigella flexneri lipopolysaccharides has shown that the O-specific side chains of these polymers consist of a primary unbranched chain of N-acetylglucosamine and rhamnose substituted with secondary side chains of glucose. Sh. flexneri Y variants which lack the glucose secondary side chains and are thus structurally identical with the primary side chains of the different serotypes, belong to one of two types namely, Y1 which consists of N-acetylglucosaminyl-(1→2)-rhamnosyl-(1→4)-rhamnose repeating units bound together by a (1→6)-linkage or Y2 which consists of N-acetylglucosaminyl-(1→3)-rhamnosyl-(1→4)-rhamnose repeating units bound together by a (1→4)-linkage. The different smooth Sh. flexneri serotypes are synthesised from these variant Y1 or Y2 precursor structures by the action of specific UDP-glucose transferases which incorporate the glucose secondary side chains in a position that characterises each serotype. The structural sequences that seem to play an immunodominant role in antigen factors I, II, III, IV, V and 7,8 are α-glucosyl-(1→4)-N-acetyl-glucosamine, α-glucosyl-(1→4)-rhamnose, O-acetyl-α-glucosyl-(1→2)-rhamnose, α-glucosyl-(1→6)-N-acetylglucosamine, α-glucosyl-(1→3)-rhamnose and α-glucosyl-(1→2)-rhamnose respectively. The group antigen 3,4 in which rhamnose is the immunodominant sugar, seems to be related to the internal rhamnosyl-(1→4)-rhamnose sequence. The serotype 6 antigen (VI) used in this study is so different structurally from its analogues that this organism is no longer regarded as a true Sh. flexneri species. From the structural relationship of the different O-antigens, X and Y variants are the consequence of enzyme defects that interrupt the biosynthesis of the complete smooth lipopolysaccharides. Y1 variants probably result from defects in the specific UDP-glucose transferases of serotypes 1a and 2a while Y2 variants probably result from defects in the analogous UDP-glucose transferases of serotypes 4a, 3a, 5 and variant X. Variant X is an intermediate stage in the biosynthesis of serotypes 3a and 5 from the precursor Y2 structure. Further evidence for the role of UDP-glucose transferases in conferring type specificity on the cryptic Y structures comes from two sources. Firstly, the type specific or T-loci of Petrovskaya that map near the lac locus are those in which glucose is the immunodominant sugar and loss of this locus by genetic recombination results in a glucose-deficient Y-type hybrid. Secondly, the structures proposed show that all phage conversions of Sh. flexneri reported to date, can be explained in terms of single enzyme changes involving specific UDP-glucose transferases. From the limited number of antigens studied, it can be predicted that the expression of type specific antigens will depend on the presence of group factors, as the former are always end group determinants which cannot be incorporated into the growing molecule before biosynthesis of the more central regions which determine group antigenicity.

Aim An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. Materials and results The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4-7 and SDS-PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti-A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver-stained gel and subjected to mass spectrometry in MALDI-TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against A siamensis is discussed in the paper. Conclusion Three of the identified proteins namely, bromodomain-containing protein, anaphase-promoting complex subunit 5 and elongation factor-2 could possibly serve as vaccine candidates against argulosis in carps.

Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

Among Escherichia coli strains received recently at the International Escherichia Centre were several which could not be determined serologically by the available test sera. Some of these strains had a possible causal relationship to the enteric disease from which they were isolated, and it was considered important to establish the hitherto unknown antigens as test antigens. This paper reports the examination of some of such strains isolated from human disease. The paper concludes with a short discussion of the present concepts of K antigens in E. coli, explaining why new K antigen numbers were not established on the basis of the strains examined.

The urological oncology section is relatively long this month, and this reflects the many high-quality manuscripts we receive. When you consider our relatively high rejection rate, you will understand just how many papers on this topic are submitted. The high quality of oncology papers is clear in this month’s section. You will also notice that all but one of them are on prostate cancer, and the reason for this is similar to that mentioned above, as this topic is, as might be expected, the most commonly submitted in this section. However, I am only too happy to reassure readers, and those primarily interested in other types of urological cancer, that the imbalance in this month’s section is not a permanent fixture. OBJECTIVE To determine the feasibility of using flow cytometry fluorescence-activated cell sorting (FACS) analysis for detecting circulating epithelial cells (CECs) in patients with hormone-refractory prostate cancer (HRPC), and to determine whether CECs can be used to predict survival in these patients. PATIENTS AND METHODS Several prognostic models that include routinely used clinical and laboratory variables for predicting survival in men with HRPC have been reported; the presence of CECs measured by reverse transcriptase-polymerase chain reaction for prostate-specific antigen (PSA) in patients with HRPC is an independent prognostic factor for survival. CECs detected by FACS analysis correlate with advanced stage and poor survival outcome. A retrospective study was conducted to assess the presence of CECs by FACS analysis in metastatic HRPC patients initiating systemic chemotherapy with a taxane-based regimen. The association between clinical variables previously described and the presence of CECs along with the effect of the magnitude of CECs on survival was calculated, in 41 patients with HRPC, all of whom had peripheral blood collected for FACS analysis. RESULTS Except for four patients, all those with metastatic HRPC had detectable CECs. Among these patients, the number of CECs/mL was correlated with age, serum PSA level and serum alkaline phosphatase (ALP). Higher serum levels of PSA and ALP predicted a poor survival outcome. Similarly, patients with ≤1.8 CECs/mL had a significantly longer survival than those with more CECs/mL (P = 0.02). With a median follow-up of 15.4 months, the median overall survival for all patients was 18.4 months. CONCLUSIONS The presence of more CECs in patients with metastatic HRPC was associated with a poorer survival outcome; levels of ≥1.8 CECs/mL were associated with a shorter survival in patients with metastatic HRPC.

This paper reviews the pathogenesis of psoriasis, in particular, the immunological cascade in psoriasis. Psoriasis is an immune-mediated skin disease where the T cell plays a pivotal role in the pathogenesis of the disease. The critical steps involved in the pathogenesis include Langerhans cell activation and maturation by antigens in the skin, activation of the T cell by mature Langerhans cells, differentiation and expansion of T cells within the lymph nodes, trafficking of activated T cells from the lymph node to the skin and the subsequent release of cytokines. These cytokines are responsible for epidermal and vascular hyperproliferation and pro-inflammatory effects. Each of these steps provides an opportunity for biological agents designed to block the psoriatic immunological cascade. This paper reviews the immunopathogenesis of psoriasis. Biologic agents in psoriasis, to be published separately, reviews the new biologic therapies that aim to selectively block the immunological steps implicated in the pathogenesis of psoriasis outlined in this paper.

The direct agglutination test (DAT) based on freeze-dried (FD) Leishmania donovani antigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%; r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%; r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL.

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.