Simple dose-escalation regimen for hydroxychloroquine-induced hypersensitivity reaction in patients with systemic lupus erythematosus enabled treatment resumption.

OBJECTIVE: This study aimed to investigate the risk factors of hydroxychloroquine (HCQ)-induced hypersensitivity in patients with systemic lupus erythematosus (SLE) and to propose a simple dose-escalation regimen in cases of mild HCQ-induced hypersensitivity. METHODS: We identified patients with SLE who started HCQ between 2009 and 2018 and cases of HCQ-induced hypersensitivity by reviewing the electronic medical charts. A simple dose-escalation regimen, starting at 40 mg/day with weekly increments of 40 mg/day to 200 mg/day, was used in patients with HCQ-induced hypersensitivity who did not require hospitalization or systemic steroid therapy. We then compared the clinical parameters of patients with and without HCQ-induced hypersensitivity and evaluated the success of our dose-escalation regimen. RESULTS: We enrolled 302 patients with SLE and identified 25 cases of HCQ-induced eruption (8.3%). The mean Naranjo score of these patients was 5.1 ± 1.4 (min 3, max 8), and all 25 patients received a 'possible' (9) or 'probable' (16) score. A mild, generalized, maculopapular rash occurred in 24 patients, and urticaria occurred in one patient at 24 days (interquartile range 15-40 days) after the start of treatment. The proportion of cyclophosphamide use, glucocorticoid consisting of prednisolone 20 mg/day or more, and initiation of SMX-TMP within 28 days were higher in patients with skin eruptions. On multivariate analysis, only cyclophosphamide use was identified as a risk factor of HCQ-induced hypersensitivity (odds ratio = 12.3 (95% confidential interval 1.4-14.3)). Thirteen of the 14 patients on the dose-escalation regimen (92.9%) tolerated continued HCQ treatment. One patient re-experienced eruptions on day 10 day after starting HCQ. CONCLUSIONS: Mild late reactions are common in HCQ-induced hypersensitivity. A simpler dose-escalation regimen enables safe and easier reintroduction of HCQ but should not be applied to patients with immediate reactions or moderate late reactions.

The use of muscle biopsy in the diagnosis of systemic vasculitis affecting small to medium-sized vessels: a prospective evaluation in Japan.

OBJECTIVES: The aim of this study was to assess the use of muscle biopsy for histopathological confirmation of small vessel vasculitis (SVV) or medium vessel vasculitis (MVV). METHOD: Muscle biopsies were performed for all consecutive cases of suspected SVV or MVV seen at Tokyo Metropolitan Tama Medical Centre between February 2012 and May 2014 except those for which a skin or renal biopsy was indicated. RESULTS: Forty-nine patients underwent muscle biopsies. All patients were followed for a minimum of 6 months. Diagnosis of SVV or MVV was made in 35 patients. An unrelated condition was diagnosed in 11 patients and no diagnoses were made in three patients. Of the 35 patients in whom SVV or MVV was diagnosed, positive muscle biopsies were obtained in 20 patients [15 microscopic polyangiitis (MPA), three polyarteritis nodosa (PAN), and two eosinophilic granulomatosis with polyangiitis (EGPA)], while other findings led to the same diagnosis in 15 (seven MPA, four GPA, three PAN, and one rheumatoid vasculitis). The sensitivity of the muscle biopsy was 57% [20/35; 95% confidence interval (CI) 50-57]. Of 13 patients presenting with peripheral neuropathy, the muscle biopsy demonstrated vasculitis in nine patients, with 75% sensitivity (9/12; 95% CI 69-75). There were no complications in the procedure apart from delayed wound healing in one patient. CONCLUSIONS: Muscle biopsy is a safe method that offers a high diagnostic yield for SVV or MVV, especially in patients with vasculitic neuropathy.

Immunochemical specificity of the combining site of murine myeloma protein CAL20 TEPC1035 reactive with dextrans.

The immunochemical specificity of the combining sites of murine myeloma protein CAL20 TEPC1035 was studied by quantitative precipitin and precipitin inhibition assays. Myeloma protein CAL20 TEPC1035 precipitated with only three dextrans, B1355S4, B1498S, and B1501S, with high proportions of alpha(1 leads to 3) linkages, but not with any other dextrans, glycogen, and pullulan. Inhibition tests with various sugars show that the combining site of myeloma protein CAL20 TEPC1035 is most complementary to panose, a trisaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc. Panose was 3.3 times more potent than a tetrasaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc alpha(1 leads to 4)DGlc and 8, 23, 42, > 42 times more active than maltose, nigerose, isomaltose, and kojibiose, respectively. These findings were paralleled by their binding properties as determined by affinity electrophoresis. The association constants (Ka) of these three dextrans to myeloma protein CAL20 TEPC1035 ranged from 3.8 X 10(3) ml/g to 5.02 X 10(3) ml/g. The association constant of inhibitor (Kia) of panose was 8.19 X 10(3) M-1. Myeloma protein CAL20 TEPC1035 is an antidextran with specificity different from those of other murine myeloma antidextrans and from human antidextrans reported previously and its combining site size is at least as large as a trisaccharide. The binding constant of methyl alpha-D-glucoside (7.2 X 10(2)) was 73% of that of panose and comparable to that of myeloma protein W3129 (9.4 X 10(2)) with a cavity-type site and 600 times lower (1.6 X 10(0)) for QUPC52 with a groove type site, indicating that the terminal nonreducing residue is held in a cavity. Inhibition data with various alpha(1 leads to 4)-linked oligosaccharides also indicate that the internal portions of these inhibitors may react directly with a portion of the combining site. These findings suggest that myeloma antidextran CAL20 TEPC1035 has a partial cavity-type combining site in which the terminal nonreducing dGlc alpha(1 leads to 6) moiety is held in a cavity with the other two sugars forming a groove. However, oligosaccharides with one or more alternating [leads to 3DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 3)DGlcl leads to] units with and without terminal nonreducing DGlc alpha(1 leads to 6) or DGLc alpha(1 leads to 3) side chains remain to be tested to determine whether structures known to be present in the three dextrans which precipitate CAL20 TEPC1035 may not prove to be more active than panose.

Combining site specificities of mouse hybridoma antibodies to dextran B1355S.

The combining sites of 12 mouse hybridoma antibodies to dextran B1355S have been characterized by quantitative precipitin assay. All antibodies preferentially bind the immunizing antigen B1355S and two other class I dextrans, B1498S and B1501S, but show substantial differences in the extents to which they cross react with class I dextrans, suggesting their clustering into five groups. Three myeloma proteins, CAL20 TEPC1035, J558, and MOPC104E, which bind dextran B1355S, each fall into a different group. There appears to be a substantial, but imperfect, correlation of DH region structure and individual idiotypic determinants with dextran binding patterns. Proteins with RY DH segments and IdI (J558) idiotypes are in groups 1 or 3, and proteins with YD DH segments and IdI (MOPC104E) idiotypes are exclusively in group 5. However, identical patterns of precipitin curves accompany very different sequences in CDR3. Antibodies of group 1, which react only with class II dextrans, differ the most in primary sequence, a finding suggesting that subsites responsible for cross reactivity with class I dextrans may be blocked and that this may be effected by side chains of different amino acids. This finding delineates a new aspect of the relationship of variability in amino acid sequence to antibody complementarity.

Recellularization of decellularized adipose tissue-derived stem cells: role of the cell-secreted extracellular matrix in cellular differentiation.

Adipose-derived stem cells (ASCs) are found in a location within the adipose tissue known as the stem cell niche. The ASCs in the niche are maintained in the quiescent state, and upon exposure to various microenvironmental triggers are prompted to undergo proliferation or differentiation. These microenvironmental triggers also modulate the extracellular matrix (ECM), which interacts with the cells through the cytoskeleton and induces downstream events inside the cells that bring about a change in cell behaviour. In response to these changes, the cells remodel the ECM, which will differ according to the type of tissue being formed by the cells. As the ECM itself plays an important role in the regulation of cellular differentiation, this study aims to explore the role of the cell-secreted ECM at various stages of differentiation of stem cells in triggering the differentiation of ASCs. To this end, the ASCs cultured in proliferation, osteogenic and adipogenic media were decellularized and the secreted ECM was characterized. Overall, it was found that osteo-differentiated ASCs produced higher amounts of collagen and glycosaminoglycans (GAG) compared to the undifferentiated and adipo-differentiated ASCs. The two types of differentiated ECMs were subsequently shown to trigger initial but not terminal differentiation of ASCs into osteo- and adipo-lineages respectively, as indicated by the upregulation of lineage specific markers. In addition, integrin subunits alpha (α) 6 and integrin beta (ß) 1 were found to be produced by ASCs cultured on cell-secreted ECM-coated substrates, suggesting that the integrins α6 and ß1 play an instrumental role in cell-ECM interactions. Taken together, this study demonstrates the importance of the ECM in cellular fate decisions and how ECM-coated substrates can potentially be used for various tissue engineering applications.

Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli.

To study the predominant binding substance for the heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli, competitive binding assays were performed with neuraminidase-treated human type B erythrocytes and 125I-labeled B subunit of LTc (LTc-B). Of all inhibitors used, the ganglioside GM1 was the most effective in inhibiting the binding of 125I-labeled LTc-B to the erythrocytes. The other gangliosides used as inhibitors, gangliosides GD1b, GD1a, GM2, GT1b and GM3, were about 24, 166, 250, 440 and at least 440 times less reactive than ganglioside GM1, respectively. With glycoproteins as inhibitors, on the other hand, hog A + H, porcine thyroglobulin and bovine salivary mucin were over 10(4) times less potent. No inhibition was obtained by other mono-, di- and polysaccharides at the highest concentrations used. These findings suggest that the predominant binding substance on neuraminidase-treated human type B erythrocytes for the LTc-B is ganglioside GM1 and that the combining site of LTc-B may be specific for the terminal disaccharide (galactose-N-acetyl-D-galactosamine)-linked portion of ganglioside GM1.

Studies on the binding substances on human erythrocytes for the heat-labile enterotoxin isolated from porcine enterotoxigenic Escherichia coli.

The binding substance for the heat-labile enterotoxin (LTp) isolated from porcine enterotoxigenic Escherichia coli was studied by competitive binding assays. The binding of 125I-labeled LTp to neuraminidase-treated human type A erythrocytes was most effectively inhibited by ganglioside GM1 among inhibitors used. Mono-, di- and polysaccharides, glycoproteins and lectins were over 10(4)-times less potent inhibitors. Similar results were also obtained in competitive binding assays with 3H-labeled ganglioside GM1 and LTp-coupled Sepharose 4B. On the other hand, hemagglutination of neuraminidase-treated human type A erythrocytes by LTp was inhibited by methyl alpha-D-galactopyranoside, galactose, melibiose and some glycoproteins, but not effectively inhibited by ganglioside GM1 at the highest concentration used. Preincubation of LTp with an appropriate amount of ganglioside GM1 resulted in much higher hemagglutination than LTp alone. Although these findings show that there may be fundamental differences between interactions with ganglioside GM1 in hemagglutination compared to interactions with ganglioside GM1 in binding, the predominant binding substance for LTp on neuraminidase-treated human type A erythrocytes is suggested to be ganglioside GM1.

Hemagglutinating and binding properties of botulinum C2 toxin.

To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition. Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II. Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones. On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes. Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose. Thyroglobulin and bovine salivary mucin were much stronger inhibitors. From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).

Immunochemical studies of mouse monoclonal antibodies to dextran B1355S--II. Combining site specificity, sequence, idiotype and affinity.

The specificities of the combining sites of 19 mouse monoclonal antibodies to dextran B1355S have been characterized immunochemically by quantitative precipitin and precipitin inhibition assays; association constants for B1355S were determined by affinity gel electrophoresis. Cross-reactive and individual idiotypes related to the BALB/c B1355S-binding myeloma proteins MOPC104E [IdI(MOPC104E)] and J558 [IdI(J558)], determined by a radioimmunoassay, and heavy-chain variable-region sequences, are presented. Antibodies to B1355S are "alpha (1----3) alpha (1----6)-specific" as determined by precipitin and precipitin inhibition assays with dextrans and oligosaccharides, respectively, containing alternating alpha (1----3) alpha (1----6) linkages compared with oligosaccharides composed solely of alpha (1----3) or alpha (1----6) linkages; all antibodies have low association constants (less than or equal to 10(5) ml/g). However, there is also considerable diversity among the proteins as seen in the five groups of different patterns of reactivity with numerous dextrans having different structures, and the variability in affinity even among antibodies showing the same fine specificity by precipitin assay. There is little observable correlation of heavy-chain variable-region amino-acid sequence with specificity or affinity; however, all proteins having D-region amino acids Tyr,Asp at positions 96,97 express the MOPC104E individual idiotype and belong to precipitin specificity group 5, the group most cross-reactive with numerous dextrans, whereas those proteins having the J558 individual idiotype, Arg,Tyr or Asn,Tyr at 96,97 are found in all five precipitin groups.

Purification of staphylococcal enterotoxins A and E by immunoaffinity chromatography using a murine monoclonal antibody with dual specificity for both of these toxins.

An immunosorbent column was prepared by coupling a murine monoclonal antibody (MAb) with dual specificity for staphylococcal enterotoxins A (SEA) and E (SEE) to Affi-Gel 10. Purification of both SEA and SEE from culture supernatants was carried out with the immunosorbent column using 0.2 M acetic acid containing 0.15 M NaCl as eluant. The yields obtained were approximately 76% for SEA and 70% for SEE. Purified SEA and SEE were found to be immunologically and electrophoretically homogeneous. Immunoaffinity chromatography using a MAb with dual specificity proved to be valuable in the purification of SEA and SEE, not only from the standpoint of percentage recovery, but also because of the degree of purity and the ease of purification.

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli.

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enterotoxigenic Escherichia coli.

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.

Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus.

To study the correlation between emetic toxin and HEp-2 vacuole activity produced by Bacillus cereus isolated from an outbreak of vomiting-type food poisoning, some properties and emetic activities of both purified HEp-2 factor (cereulide) and partially purified factor to rhesus monkeys were determined. The results indicate that both cereulide and partially purified factor were very stable to digestion with proteolytic enzymes, different pH, and heating. Vomiting was induced in the rhesus monkeys orally administered with both substances. From these findings, cereulide (or HEp-2 vacuole factor) is strongly suggested to be an emetic toxin itself.

Murine monoclonal antibodies reactive with staphylococcal enterotoxins A, B, C2, D, and E.

By fusion of mouse spleen cells immunized with five different staphylococcal enterotoxins (SEA, SEB, SEC2, SED, and SEE) with myeloma cells, we obtained 15 hybridomas producing monoclonal antibodies (mAbs). Four mAbs were reactive with both SEA and SEE, whereas 8 mAbs were reactive with SEB and SEC2. One mAb reacted with SEA, SED, and SEE. The other two mAbs were found to be reactive with all five serotypes of SEs. The mAbs specific for five serotypes of SEs were found to be most reactive with SED, reactive with SEA, and slightly less reactive with SEB, SEC2, and SEE. Those mAbs with specificities for all serotypes of SEs may be valuable to prepare immunoadsorbent(s) for isolation of SEs and to detect SEs in foods and clinical specimens involved in outbreaks of staphylococcal food poisoning.

Improved methods for purification of an enterotoxin produced by Bacillus cereus.

An enterotoxin (ET) produced by Bacillus cereus was purified by ammonium sulfate precipitation followed by chromatography on SP-Sephadex C-25, chromatofocusing, and gel filtration on Sephacryl S-300. Purified ET was electrophoretically homogeneous with a molecular mass of 45,000 and with an isoelectric point of 5.5. It showed vascular permeability activity in rabbits, was lethal to mice, and caused fluid accumulation in mouse ligated intestinal loops. It showed cytotoxicity to Vero cells, but did not show any hemolytic or phospholipase C activities. These biological activities were found to be easily inactivated by heating at 56 degrees C for 5 min, by digestion with trypsin and pepsin, and by exposure to pH below 3.0 or higher than 11.

Partial nucleotide sequencing of the NS3/helicase region of hepatitis G virus to prove vertical transmission.

To study non-parental transmission of hepatitis G virus and/or GB virus C (HGV/GBV-C), we sequenced and compared the NS3/helicase region of the virus for five HGV/GBV-C RNA-positive mothers and their 11 children who had experienced neither blood transfusion nor overt hepatitis and were negative for HBV, HCV and HIV, except in one mother coinfected with HCV. The nucleotide sequences of the familial HGV/GBV-C isolates showed high similarity of 99-100% (mean 99.8%, 100% at the deduced amino acid level) between mother and her child(ren) in each family. These findings strongly suggest the spontaneous occurrence of mother-to-child transmission of HGV/GBV-C as reported previously. They also suggest that nucleotide sequence analysis on the NS3/helicase region of HGV/GBV-C may be a useful tool to study HGV/GBV-C transmission.

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A.

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.

Studies on the functional site on staphylococcal enterotoxin A responsible for production of murine gamma interferon.

To identify the functional region(s) associated with induction of gamma interferon on the staphylococcal enterotoxin A molecule, native staphylococcal enterotoxin A molecules and 12 various synthetic peptides corresponding to different regions of entire staphylococcal enterotoxin A were compared to induce gamma interferon production in murine spleen cells. The native staphylococcal enterotoxin A molecule induced gamma interferon production, whereas all of the 12 synthetic peptides did not. Pre-treatment of the murine spleen cells with synthetic peptide A-9 (corresponding to amino acid residues 161-180) significantly inhibited the staphylococcal enterotoxin A-induced gamma interferon production, whereas those with other synthetic peptides did not. When native staphylococcal enterotoxin A was pre-treated with either anti-staphylococcal enterotoxin A serum or anti-peptide sera, anti-staphylococcal enterotoxin A serum and antisera to peptides A-1 (1-20), A-7 (121-140), A-8 (141-160), A-9 (161-180) and A-10 (181-200) inhibited the staphylococcal enterotoxin A-induced gamma interferon production. From these findings, the amino acid residues 161-180 on the staphylococcal enterotoxin A molecule may be an essential region for murine gamma interferon production. Furthermore, the neutralizing epitopes may be also located on regions of amino acid residues 1-20, 121-140, 141-160 and 181-200 on the staphylococcal enterotoxin A molecule.

Frequency of Shiga toxin-producing Escherichia coli in cattle at a breeding farm and at a slaughterhouse in Japan.

To investigate the frequency of Shiga toxin-producing Escherichia coli (STEC) infected calves at a breeding farm and cattle at a slaughterhouse in Tohoku area of Japan, the polymerase chain reaction (PCR) was used for detection of genes for Shiga toxin(s). The fecal samples from a total of 204 calves and 306 cattle were examined. The prevalence rates in calves less than 2 months of age, cattle 2-8 months of age, and adults greater than 1 year of age were 39.4, 78.9, and 40.8%, respectively. Detection frequency of STEC in the fecal specimens from calves aged 0-8 months was not different among the breeds of cattle (Holstein: H, Japanese black cattle: B, and F1: HxB). On the other hand, for calves over 12 months of age, the frequency of STEC in Japanese black cattle and F1 were significantly higher than in Holstein cattle. Serogroups of STEC usually identified in human cases of food poisoning (O157, O26, and O111) were not frequently found in the feces of the cattle.

Changes in concentrations of serum amyloid A protein, alpha 1-acid glycoprotein, haptoglobin, and C-reactive protein in feline sera due to induced inflammation and surgery.

To identify candidates for feline acute phase proteins, the concentrations of serum amyloid A protein (SAA), alpha 1-acid glycoprotein (alpha 1-AG), C-reactive protein (CRP), and haptoglobin (Hp) were measured in sera isolated from clinically normal and hospitalized (or diseased) cats, from cats with experimentally induced inflammation, and cats subjected to surgery for urinary diversion. Measurements were made by sandwich enzyme-linked immunosorbent assay and single radial immunodiffusion. The concentrations of SAA, alpha 1-AG, and Hp in sera from hospitalized cats were 7-11 times higher than in clinically normal cats. Similar results were obtained for the concentrations of SAA, alpha 1-AG, and Hp in cats with induced inflammation and cats subjected to surgery. By contrast, the serum concentration of feline CRP did not change significantly between clinically normal cats and hospitalized cats or inflammation-induced or post-surgery cats. Feline SAA concentration was found to increase earliest, with alpha 1-AG and Hp beginning to increase thereafter. From these results, feline SAA is concluded to be an acute phase reactant at the early stage of inflammation.