Jan Fohlman

Isolation and characterization of a lethal myotoxic phospholipase A from the venom of the common sea snake Enhydrina schistosa causing myoglobinuria in mice.

Abstract A strongly myotoxic phospholipase A was isolated from the venom of Enhydrina schistosa by gel filtration on Sephadex G-75 and ion exchange chromatography on Bio-Rex 70. The myotoxin is a basic protein containing 120 amino acid residues and seven disulfide bridges. The formula weight is 13,500. The release of myoglobin diagnostic of muscle necrosis was monitored by placing injected mice on sheets of white paper. Affected mice stained the paper red. The myotoxin has an acute ld 50 of 110 μg/kg and causes myoglobinuria at doses down to 30 μg/kg. The molecule lacks trytophan, indicating that this amino acid is not required either for toxicity or catalytic activity. A single histidine residue was modified by reaction with p -bromophenacyl bromide, resulting in > 99% loss of enzymatic and toxic activities. The N-terminal sequence shows homology with nontoxic and neurotoxic phospholipases A 2 of vertebrate origin. The myotoxicity may be of greater clinical importance than the curarimimetic toxins that have received so much attention heretofore.

Coxsackie B virus IgM in children at onset of type 1 (insulin-dependent) diabetes mellitus: evidence for IgM induction by a recent or current infection.

SummaryThirty-five children with newly-diagnosed Type 1 (insulin-dependent) diabetes mellitus and their 47 siblings were investigated for the presence of IgM antibodies to Coxsackie B virus serotypes 1–5 (CBV1-5) with the aid of μ-antibody-capture radioimmunoassays. When a high cut-off value was used, 16 (46%) diabetic children and 16 (34%) siblings showed CBV-IgM. Of the siblings of diabetic patients positive for CBV-IgM, 11 (44%) were CBV-IgM-positive; the corresponding figure for the siblings of negative patients was five (26%). With a lower cut-off value, leading to a “borderline titre”, the frequency of IgM positivity increased in both the patients and siblings. When the borderline titres were included, the number of IgM-positive patients was 19 (54%) and the corresponding number of siblings was 29 (62%). Of the siblings of positive patients, 27 (93 %) showed CBV-IgM, and of the siblings of the negative patients, two (11 %) were CBV-IgM-positive. Sixteen (89 %) siblings of IgM-negative patients remained negative. Regarding the serotypes of CB V to which IgM was directed, CBV 4 was most frequent, followed by serotypes CBV 3, CBV 2, CBV 5 and CBV 1. There was a striking similarity between the individual diabetic child and his or her sibling(s) concerning this specificity of IgM. It is concluded that within most families with a newly-diagnosed diabetic child positive for CBV-IgM the same serotype(s) of the virus circulates and that the intrafamilial spread of virus is considerable. The results strongly indicate that the IgM detected was CBV-specific and caused by a recent or current CBV infection. It is highly probable that the same strain of virus was present in different members of the same family. Therefore, if diabetogenic CBV strains do in fact exist, additional factors must be of importance for the development of Type 1 diabetes in children infected with such a CBV strain but remaining non-diabetic.

Effects of methyl mercury on cytokines, inflammation and virus clearance in a common infection (Coxsackie B3 myocarditis)

A myocarditic coxsackievirus B3 (CB3) infection in Balb/c mice was used to investigate the effects of 12 weeks of methyl mercury (MeHg) exposure (3.69 mg/g diet) on inflammatory heart lesions, virus in the heart, the cytokine response, i.e. cachectin/TNF-alpha and gamma-interferon (IFN-gamma) levels in plasma, and on disease complications and mortality. This dose of MeHg did not influence mortality in this infection model. The inflammatory and necrotic lesions in the ventricular myocardium 7 days after the inoculation covered 2.2% of the tissue section area in infected control mice. This damage was increased (n.s.) by 50% (to 3.3% of the tissue section area) in MeHg-treated mice. The response pattern of lymphocyte subsets in situ in myocardial inflammatory lesions was corroborated using an immune histological technique. MeHg treatment tended to increase (2.2-fold, n.s.) the number of Mac 2+ cells (macrophages) in the heart muscle in this infection. Plasma levels of both TNF-alpha and IFN-gamma increased on day 3 of the infection in MeHg-treated as well as in non-MeHg-treated mice, but the mean IFN-gamma response was more pronounced in the MeHg-treated mice. On day 7 of the infection, when most animals still showed clinical signs of disease, cytokine levels were back to normal. MeHg-exposure in non-infected mice did not affect cytokine levels. In situ hybridization of virus RNA in myocardial tissue showed remaining virus in those mice who had the lowest plasma IFN-gamma levels. A 20% increased (P < 0.05) lymphoproliferative response to the T cell mitogen Con A was observed as a result of the MeHg treatment. Even heart tissue lesions and virus persistence tended to be influenced by MeHg in a direction compatible with the development of chronic disease.

Adsorption phenomena on sephacryl® S-200 superfine

Abstract The adsorption properties of Sephacryl S-200 are dependent upon the pH of the eluent buffer employed at the net surface charge of the solutes under investigation. At pH 3.5 the gel adsorbs acidic, neutral and basic proteins. By raising the pH of the eluent to 5.5 all adsorbed material can be desorbed from the gel. At pH 5.5 the gel showed no tendency to adsorb proteins of any surface charge. At this pH the gel acts as a passive molecular sieve. At pH 8 the gel begins to behave as a cation exchanger and strongly adsorbs any protein that is positively charged at this pH. Acidic or neutral proteins are eluted unretarded. These cation-exchange properties can be eliminated by including at least 0.2 M NaCl in the eluent buffer. At low ionic strength of the eluent buffer, DNA, tRNA and rRNA are adsorbed on to the gel at pH 3.5 or 5.5 but are completely desorbed by stepwise elution with 0.5 M NaCl in equilibrating buffer. The HETP values calculated for several solutes indicate that Sephacryl S-200 gives higher resolution and less zone-spreading than does Sephadex G-200.

High-pressure liquid chromatographic identification of phenylthiohydantoin derivatives of all twenty common amino acids.

Abstract Phenylthiohydantoin (PTH) derivatives of all 20 common amino acids can be separated by high-pressure liquid chromatography. By using a Waters reversed-phase C18 column eluted with a concave ethanol gradient in ammonium acetate, pH 5.1, all PTH derivatives were eluted in less than 30 min. The NH2-terminal amino acid sequence of the human retinolbinding protein could unambiguosly be established for the first 40 residues. Likewise, HLA-DR antigens biosynthetically labeled with [3H]tyrosine and [3H]phenylalanine were subjected to automatic sequential degradation. Labeled PTH-amino acids were easily identified by the described chromatographic procedure.

Vaccination of Balb/c mice against enteroviral mediated myocarditis

A non-virulent strain of Coxsackie B3 (CB3) virus was used to produce a subunit vaccine. It contains the capsid proteins VP1, 2, 3 and probably 4 and can be made RNA-free. It is based on the ISCOM technology ensuring non-toxic properties and good adjuvant effect. Vaccinated animals at doses above 16 ng were completely protected from mortality when challenged with a myocarditic strain of CB3. Histologically no inflammatory lesions were found in the heart. This was corroborated using immune histological techniques with monoclonal antibodies against lymphocyte subsets. Even at a dose of 0.16 ng a delayed mortality was observed. Neutralizing antibody titres rose to 512, thus ensuring a circulating level well above that considered protective. It is suggested that vaccination might be a possible way of prophylaxis for myocarditis and even dilated cardiomyopathy, the latter presently being the chief cause of heart transplantation. By persistence or triggering of autoimmune phenomena Coxsackie virus is incriminated as the first step in pathogenesis.

Trace element changes in the pancreas during viral infection in mice.

Introduction The trigger for some cases of juvenile diabetes has been suggested to be an interaction between a virus and various trace elements. Infection with human coxsackievirus B3 (CB3) in the murine model results in viral replication and inflammation in the pancreas. Aim To determine how infection affects the trace element balance in the pancreas. Methodology Concentrations of the following trace elements were measured in the serum and pancreas during the early phase (days 1 and 3) of CB3 infection in female Balb/c mice: aluminium, arsenic, cadmium (Cd), calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), mercury (Hg), selenium, silver, vanadium (V), and zinc (Zn). The trace element concentrations were measured through inductively coupled plasma mass spectrometry. The histopathology was established by hematoxylin–eosin techniques and immunohistochemical staining of both CD4 and CD8 cells of the pancreas. Results Infected mice developed expected clinical signs of disease. The only changes at day 1 occurred in the serum, with a pronounced decrease in the Zn concentration and a small increase in the V concentration. At day 3, concentrations of several trace elements, including Cu, Zn, Fe, Ca, V, and Mn, showed pronounced changes in both the serum and the pancreas. Ca, Cu, Mg, Mn, and V, but none of the potentially toxic elements, accumulated in the pancreas. Cu and V concentrations increased in the serum as well. Conclusion Several trace element changes, preceding the development of pancreatitis, occurred in the pancreas in this viral infection, the exact pathogenic interpretation of which warrants further studies.

Trace element distribution in heart tissue sections studied by nuclear microscopy is changed in Coxsackie virus B3 myocarditis in methyl mercury-exposed mice.

Methyl mercury (MeHg) has been shown to change Coxsackie virus type B3 (CB3) myocarditis in a direction compatible with the development of chronic disease. Murine models of CB3 myocarditis closely mimic the pathogenesis in humans. There are also indications that metals, such as mercury, and trace elements may interact and adversely affect viral replication and development of inflammatory lesions. The effects of low-dose MeHg exposure on myocardial trace element distribution, as determined by means of nuclear microscopy, was studied in CB3 myocarditis. Balb/c mice were fed a MeHg-containing diet (3.9 mg/kg diet) for 12 wk prior to infection. Areas of inflammatory lesions in the myocardium were identified by traditional histologic examination, and serial tissue sections in these selected areas were used for immune histology (macrophages), in situ hybridization of virus genomes, and nuclear microscopy of tissue trace element distribution. Areas with no inflammation or virus were compared with areas of ongoing inflammation and viral replication. In the inflammatory lesions of MeHg-exposed mice as compared to nonexposed mice, the myocardial contents of calcium (Ca), manganese (Mn), and iron (Fe) were significantly increased, whereas the zinc (Zn) content was decreased. The increased Ca and decreased Zn contents in the inflamed heart may partly explain a more severe disease in MeHg-exposed individuals. Although not significant in the present study, with a limited number of mice, the inflammatory and necrotic lesions in the ventricular myocardium on d 7 of the infection was increased by 50% (from 2.2% to 3.3% of the tissue section area) in MeHg-exposed mice and, also, there was a tendency of increased persistence of virus with MeHg exposure. No increased MeHg uptake, either in the inflammatory lesions or in the areas of noninflamed heart tissue in infected mice, could be detected. The present results indicate that a “competition” exists between potentially toxic heavy metals from the environment/diet and important trace elements in the body and that a disturbed trace element balance adversely influences the development of pathophysiologic changes in inflammatory heart disease.

Some Phylogenetical Aspects on the Occurrence of Somatostatin in the Gastro-Entero-Pancreatic Endocrine System

Rodioimmunoassayable somatostatin (SRIF) was found in acid ethanol extracts from various parts of the gastro-entero-pancreatic (GEP) endocrine system in reptiles, amphibians, teleost bony fish, cartilaginous fish, and jawless fish, as well as in a deuterostomian invertebrate, the tunicate, Ciona intestinalis. The cellular sites could, as a rule, be easily visualized light-microscopically by the peroxidase-anti-peroxidase (PAP) immunocytochemical procedure, using guinea-pig and rabbit antisera against synthetic SRIF. The standard Hellerström-Hellman technique, used to detect argyrophi SRIF-storing D cells, failed to visualize the SRIF cells in teh GEP endocrine system of the tumicate and of the jaw-less fish. Moreover, the results comfirmed the previous description that this technique only exceptionally (and sometimes only after further modifications) gave positive results when applied to the GEP endocrine system of bony fish, amphibians, and reptiles. In cartilaginous fish, however, it worked adequately and confirmed the radio-immunological and immunocytochemical observations. In the mucosa of the alimentary tract and in the parenchyma of its associated glands of one echinoderm and two pelecypod molluscs and one crustacean arthropod no sgns of the occurrence of SRIF-storing cells were observed using the three correlated procedures. In several of these tissues, signs of the occurrence of insulin-producing cells had perviously been observed. Thus, SRIF seems to appear at a later evolutionary stage than insulin. The principal islets (Brockmann corpusles) of the marine teleost fish, Cottus scorpius, had the highest concentrations of radioimmunoassayable SRIF of all the GEP organs and tissues investigated, viz. about 200 ng/mg wet weight. Nevertheless, it was only 1/5 of the actual insulin content.

An Incidence Peak of Juvenile Diabetes. Relation to Coxsackie B Virus Immune Response

ABSTRACT. All new eases of insulin dependent diabetes mellitus (IDDM) in children below 15 years of age were recorded prospectively during a 21–year period 1964–1984 in a defined uptake area with a relatively constant child population. The total number of children recorded was 222–111 boys and 111 girls. The number of new cases varied between 4 cases in 1968 and 20 in 1984; in 1983 seventeen new caw were recorded. Specific IgM antibodies against Coxsackie B virus (CBV), types 1–5 were measured by a reverse radioimmunoassay (RIA) technique in 24 consecutive patients collected during the period March 1982–January 1984, some of whom represented the recent period of a very high incidence of diabetes. Sixteen patients (67%) exhibited CBV IgM responses, strongly suggesting a current or recent CBV infection. The titres declined during the first few months of diabetes and seemed to be absent after the first half‐year period. Among age‐matched non‐diabetic children scheduled for elective procedures during the same period, no cases with CBV‐IgM antinbodies were detected. Only three of the 16 IgM‐RIA‐positive patients showed a signircant rise in the neutralising antibody titre against the same Coxsackie B type. It is concluded that CBV may play a pathogenetic role in induction of IDDM, and possibly more frequently so during periods with a high incidence of diabetes, at least in children below 15 years of age.