Lactobacillus plantarum NCIMB8826 ameliorates inflammation of colon and skin in human APOC1 transgenic mice.

Genetic predisposition and environmental factors, including the gut microbiota, have been suggested as major factors in the development and progression of atopic dermatitis. Hyperlipidemic human APOC1(+/+) transgenic mice display many features of human atopic dermatitis, such as scaling, lichenification, excoriations, and pruritus, along with a disturbed skin barrier function. Cytokine analysis of serum shows an increase of various pro-inflammatory cytokines, including interleukin (IL)-12p40, IL-6, and IL-1α, but lower levels of interferon-γ. These mice also display aspects of colitis evident from macroscopic and histological abnormalities. Genome-wide transcriptome analysis of the intestine shows up-regulation of several genes associated with mast cells and eosinophils and this observation was confirmed by demonstrating increased numbers of IgE(+) and FcRε(+) mast cells in the colon and in the skin. Oral treatment with Lactobacillus plantarum NCIMB8826 resulted in decreased numbers of mast cells in the colon. Moreover, this L. plantarum strain ameliorated skin pathology, evident from improved skin barrier integrity, absence of skin thickening, and less excoriations. These results suggest that modulation of intestinal immune homeostasis contributes to the suppression of atopic dermatitis.

Use of combined oral narcotic and benzodiazepine for control of pain associated with bone marrow examination.

BACKGROUND: Bone marrow aspirate and biopsy is universally recognized as being painful. Few descriptions of effective analgesia or premedication for this procedure exist. In this study, we assessed an oral narcotic and benzodiazepine combination in controlling pain associated with bone marrow examination. METHODS: Twenty-four consecutive ambulatory, adult patients referred for bone marrow examination received oral medications 90 minutes before the scheduled procedure. Patients reported perceived pain, using both Likert numerical and "Faces Pain Rating Scale," immediately after bone marrow examination and within 1 week after the procedure. Physicians' and nurses' evaluations of patient tolerance and the patients' memories of the aspiration and biopsy were recorded. RESULTS: Two thirds (66%) of the respondents reported none or only mild pain (3 or less on a scale of 1 to 10). Memory of the procedure was vague or nonexistent in approximately half of the patients. There were no complications of biopsies or premedication. CONCLUSIONS: Premedication with oral narcotic and benzodiazepine is effective in preventing or lessening pain associated with bone marrow examination in adults. Premedication induces amnesia for some or most of the procedure in about half of the patients.

EBV-induced expression and HLA-DR-restricted presentation by human B cells of alpha B-crystallin, a candidate autoantigen in multiple sclerosis.

The development of multiple sclerosis is most likely influenced by autoimmune responses to central nervous system myelin proteins as well as by infections with common viruses such as EBV and human herpesvirus-6. However, much remains to be established on how these factors interact. In this study, we show that upon EBV infection, human B cells start to express alpha B-crystallin, a small stress protein that was identified previously as an immunodominant Ag of CNS myelin in multiple sclerosis patients. EBV-induced expression of alpha B-crystallin in B cells leads to HLA-DR-restricted presentation of the protein and to activation of proinflammatory alpha B-crystallin-specific Th cells. While alpha B-crystallin is present in EBV-infected human B cells, the protein is absent from human lymphoid tissues under normal conditions. This is in sharp contrast to other stress proteins such as heat-shock protein (hsp)27 and hsp60 that are ubiquitously expressed in these tissues. In addition, the absence of alpha B-crystallin from lymphoid tissues in humans is unique as compared with other mammals. All other species examined, including rodents, sheep, and primates, showed constitutive expression of alpha B-crystallin in secondary lymphoid tissues and sometimes even in the thymus. Since constitutive lymphoid expression most likely results in immunologic tolerance, such a state of tolerance to alpha B-crystallin can be expected for all of these species, but not for humans. When taken together, our data provide evidence for a novel mechanism by which common viral infections can trigger myelin-directed autoimmunity in a way that is unique for humans.

T cells discriminate between differentially phosphorylated forms of alphaB-crystallin, a major central nervous system myelin antigen.

Factors such as developmental stage or physiological and infectious stress may change patterns of post-translational protein modification. In order to determine whether such regulated types of modification may influence T cell responsiveness to self proteins we examined the T cell response of SJL (H-2s) mice to alphaB-crystallin, a small heat shock protein that can exist in differentially phosphorylated forms. Epitope mapping revealed the presence of two T cell epitopes that are presented by I-As. One major epitope including residues 41-56 contains an amino acid residue (Ser45) that can be phosphorylated as the result of aging or stress. Accordingly, T cells from SJL mice discriminate between preparations of alphaB-crystallin that differ in their extent of phosphorylation at the level of whole protein as well as at the level of determinant-specific responses. Phosphorylation at Ser45 does not prevent binding of the peptide 41-56 to I-As and computer-assisted modelling of the peptide-MHC complex suggests that the phosphate group of the bound peptide extends outwards from the peptide-binding cleft and may thus be available for direct contact with TCR. Together, our data provide evidence that stress-inducible phosphorylation of alphaB-crystallin creates neo-determinants for T cells and, therefore, may contribute to the breakdown of peripheral tolerance to this self protein.

Yeast expressing hepatitis B virus surface antigen determinants on its surface: implications for a possible oral vaccine.

The two major hydrophilic regions of the hepatitis B virus surface antigen (HBsAg) have been expressed in the outer mannoprotein layer of the cell wall of "Bakers Yeast", Saccharomyces cerevisiae, by fusing them between the yeast invertase signal sequence and the yeast alpha-agglutinin carboxyterminal cell wall anchoring sequence. The fusion protein contained most of the preS sequences, including the hepatocyte receptor, and part of the S sequence including the "a" determinant, and was expressed from multiple genomic copies (MIRY) using the constitutive PCK promoter. Immunofluorescence studies showed that the fusion protein was detectable at the cell surface and was stably expressed at a relatively high level. Intraperitoneal immunization of mice revealed a very weak response against the S region, and a high response against yeast itself. It is proposed that increasing the amount of the antigen and reducing the number of native cell wall proteins, might lead to a yeast that is usable as a safe and cheap live oral vaccine.

The involvement of specific anti myelin basic protein antibody-forming cells in multiple sclerosis immunopathology.

Irrespective of the large body of literature on the putative role of antibodies in the development of multiple sclerosis (MS), the detection of specific antibody-forming B cells (AFCs) in the central nervous system (CNS) tissues has not been described. In this study we show that autoantigen-specific AFCs can be found in CSN tissue sections of MS patients. Applying a newly developed myelin basic protein (MBP)-enzyme conjugate technique, we have detected MBP-specific AFCs in autopsy periventricular white matter and cerebellum tissue sections of MS patients. We demonstrated the presence of MBP-specific AFCs in CNS tissue sections in five out of 12 MS patients. No MBP-specific AFCs were detected in CNS tissue sections of 11 patients with other neurological diseases, such as Parkinson's and Alzheimer's disease, or in brain tissue sections of eight deceased persons without neurological diseases. In MS patients, anti-MBP AFCs were present in brain tissue sections both with and without plaques. The proportion of MBP-specific AFCs in some of the MS patient brain tissues reached over 50% of all AFCs. The high relative frequency of the anti-MBP AFCs and their localization in periventricular white matter and cerebellum of MS patients only, suggests that anti-MBP AFCs represent a cell population, which could play an important role in MS immunopathology.

Immunohistochemical detection of co-localizing cytokine and antibody producing cells in the extrafollicular area of human palatine tonsils.

In vitro experiments have documented the role of cytokines in the regulation of the human humoral immune response. Which cytokines are operative in vivo and in which lymphoid compartment interactions between cytokine-producing T cells and antibody-forming B cells occur is still unclear. For that reason we studied human tonsils using immunohistochemical techniques. In tissue sections from tonsils in a resting stage after recurrent tonsillitis we observed cells producing IL-1 alpha and tumour necrosis factor-alpha (TNF-alpha) which were exclusively localized in the mantle zone of the follicle and in the extrafollicular area. Furthermore, a high frequency of interferon-gamma (IFN-gamma)-producing cells was detected in the extrafollicular area, but not inside the follicles. Occasional IL-2- and IL-4-producing cells were found in the extrafollicular area. Immunohistochemical detection of antibody isotypes revealed that B cells, IgM-membrane-positive, were localized inside the follicles and mantle zones, whereas IgD-membrane-positive cells were mainly found in the mantle zones of secondary follicles. In contrast, plasma cells producing IgG1-4 and IgA1-2 were found in the extrafollicular area. No IgD and IgE antibody-forming cells were detected in tonsils, whereas IgM antibody-forming cells were detected in the extrafollicular area. The co-localization of cytokine-producing cells and antibody-forming cells in human tonsil suggests that T-B cell interactions, required for B cell differentiation and isotype switching, take place in the extrafollicular area.

Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

Detection of genetic variants of alpha 1-antitrypsin with site-specific monoclonal antibodies.

The serum protein alpha 1-antitrypsin (alpha 1-AT) serves as the major inhibitor of neutrophil elastase. The most common allele of the alpha 1-AT gene is designated as PiM. The Z mutation is a single-base substitution of the normal M allele, causing a Glu----Lys change at position 342 in the molecule. The ZZ phenotype is associated with a severe deficiency of alpha 1-AT, serum concentrations of the protein being 10% of normal. Individuals with an alpha 1-AT deficiency are at an increased risk of developing emphysema. To generate antibodies that specifically detect the 342 position in the context of the flanking sequences, we synthesized several peptides that included the 342 position for both the M and the Z variant. Immunization with variant-specific peptide-carrier conjugates elicited alpha 1-AT variant-specific responses, as determined in a direct enzyme-linked immunoassay. Monoclonal antibodies (MAbs) were selected with different specificity for the 342 region: MAbs F43 recognize only the alpha 1-AT sequence with 342Glu, i.e., all variant proteins that are non-Z, either from hetero- or homozygous individuals; MAbs F50 recognize only the sequence with 342Lys, i.e., all Z-variant proteins in ZZ or heterozygous individuals; MAbs F46 recognize alpha 1-AT with either 342Lys or 342Glu, all variant proteins with sequences as in the peptides used. Z homo- and heterozygotes were detected with our MAbs in a rapid and simple immunoblot assay. Other variants (M, S, and F) can also be assigned on the basis of the electrophoretic pattern. This sensitive detection method is very easy, rapid, and straightforward and provides a powerful tool for diagnosis of the alpha 1-AT deficiencies, allowing early treatment (augmentation of alpha 1-AT) and proper advice on lifestyle practices.

An improved conjugation method for controlled covalent coupling of synthetic peptides to proteins using glutaraldehyde in a dialysis method.

Controlled and efficient conjugation of synthetic peptides to proteins, for use in immunization or in assay procedures, is a prerequisite for the immunological applications of synthetic peptides. This study describes a new method of conjugating synthetic peptides to proteins in such a way that no homopolymers of synthetic peptides or proteins occur. To achieve this, the protein is first activated with glutaraldehyde and subsequently excess glutaraldehyde is removed. Then coupling of the synthetic peptide to the activated protein occurs while subsequently the surplus reactive glutaraldehyde groups on the protein are blocked with lysine. Excess free peptide and lysine is then removed by dialysis. This improvement not only results in better defined conjugates when compared to classical glutaraldehyde coupling, but also in the consumption of smaller amounts of synthetic peptide during conjugate formation. When used for immunization we obtained similar and sometimes even better responses with the glutaraldehyde based conjugates than with succinimidyl (MBS) conjugates of the same peptides. The performance of the modified conjugates in ELISA procedures, immunization and immunocytochemistry suggests that they are superior to conjugates formed by classical glutaraldehyde coupling.

A novel carbodiimide coupling method for synthetic peptides. Enhanced anti-peptide antibody responses.

Coupling of peptides to immunogenic protein carriers is required for the generation of anti-peptide antibody responses. Carbodiimides are hetero bi-functional coupling reagents that are utilized for coupling reactions through carboxyl and amino groups. The procedures generally used for carbodiimide coupling of peptides and proteins result in conjugates which generate immunodominant antibody responses directed against the neodeterminants on the carrier protein. These determinants are induced by the reaction of carrier and/or peptide with the coupling agent. We have investigated the potential inhibiting effect of an imidazole intermediary on the formation of unwanted neodeterminants during carbodiimide coupling. The serum antibody responses elicited with the peptide-protein conjugates produced were evaluated in ELISA. We have modified and improved the coupling with a watersoluble carbodiimide (EDC) in such a way that a high response to the coupled peptide is obtained in association with negligible levels of anti-neodeterminant antibodies.

Antibodies to a short synthetic peptide related to the hinge segment of human IgG3 recognizes thermally or fixative induced conformational changes in the human IgG3 molecule.

A synthetic decapeptide (SP) was used to produce a murine monoclonal antibody specific for the human IgG3 molecule. Recognition of the IgG3 determinant is heat- and fixation-sensitive in ELISA and immunoenzyme cytology, respectively. The antibody specifically recognizes a sequence from the hinge region of IgG3, but only when subtle alterations in the conformation are induced by mild heating (greater than 40 degrees) and subsequent stabilization by means of electrostatic interactions in solid-phase assays or by fixation with formalin acetic acid mercury chloride. The structure of the human IgG3 molecule is especially sensitive to microenvironmental influences, as can be concluded from its behaviour under various physicochemical conditions. To this, we add that the immunogenic determinants in the structure of relatively flexible parts of this protein can be severely altered by the routine application of fixation methods. It is shown that these changes can also be of importance in the recognition of antigen by monoclonal antibodies.

Marginal zone of the murine spleen in autotransplants: functional and histological observations in the response against a thymus-independent type 2 antigen.

Splenic tissue from mice was autotransplanted; after initial necrosis, a rapid restoration of implants into a structure histologically indistinguishable from splenic tissue was observed. The development of the marginal zone in these autotransplants, as determined with monoclonal antibodies against different splenic cell types and routine histological stains, was compared with the local and systemic response against a thymus-independent (TI) type 2 antigen. Full restoration of time course and peak of anti-trinitrophenyl (TNP) serum titres against TNP-Ficoll was observed at 4 weeks after autotransplantation. Anti-TNP antibody-forming cells were observed in subnormal and normal numbers in 2- and 4-week old autotransplants, respectively. The appearance of normal numbers of antibody-forming cells, and the restoration of antibody titres at week 4 correlated with the return of newly formed B cells in a normal marginal zone. An unexpected observation was that marginal zone macrophages did not return until 10 weeks after transplantation, thereby making the necessity for these cells in the normal TI-2 response unlikely. We conclude that normal anti-TI-2 responses (onset and peak titres) can be restored by autotransplantation of splenic tissue. B cells and marginal zone organization are responsible for this response, for which marginal zone macrophages seem expendable. The partial protection against overwhelming post-splenectomy infections, given by autotransplants, can thus be explained by restorative capabilities of these implants on antigen presentation and antibody formation against TI-2 antigens, and not by an increase (compared with splenectomized individuals) of phagocytosis by marginal zone macrophages.

IgG subclass distribution in juvenile human tonsil: IgG3 and IgG4 results of specific antibody production using synthetic peptides.

A decapeptide with a sequence corresponding to a part of the hinge region of human IgG3 was used to prepare a mouse monoclonal antibody (Mab 330-2.2). The Mab recognized IgG3 in ELISA only when the IgG3 was denatured by mild heat. Mab specificity in immunohistochemistry was calibrated with a number of reference Mabs obtained through a WHO/IUIS collaborative study. Finally, Mab 330-2.2 was used in conjunction with a set of other isotype specific Mabs to study the (sub)class distribution of cytoplasmic Ig containing cells in palatine tonsil. IgG3 within the IgG class was strongly over-represented in our tonsil material if compared to the representation of IgG3 in serum.