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Reunanen A, Roivainen M, Kleemola M, Saikku P, Leinonen M, Hovi T, Knekt P, Leino A, Aromaa A (National Public Health Institute, Helsinki and Oulu; Social Insurance Institution, Turku; University Hospital, Turku, Finland). Enterovirus, mycoplasma and other infections as predictors for myocardial infarction. Journal of Intern Med 2002; 252: 421–429. Objectives. To study antibodies against five infectious agents for their prediction of major coronary events in men with and without evidence of coronary heart disease at baseline. Design. A case–control study nested within a prospective population study. Subjects. The study cases included 441 men 45–64 years old with nonfatal myocardial infarction or coronary death within a mean follow-up time of 10 years. A total of 165 men had already signs of heart disease at baseline, whilst 276 were apparently healthy at the beginning of the study. Two controls for each case were matched for age, heart disease status and place of residence. Antibodies against enterovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, cytomegalovirus and adenovirus were determined. Results. Men without reported baseline heart disease, but not those with heart disease, showing the highest quartile of antibodies to enterovirus and mycoplasma or increased levels of immune complex-bound antibodies to chlamydia had a significantly higher risk of coronary events than men with lower level of antibodies. The increased risk demonstrated in men with high levels of antibodies to enterovirus and mycoplasma remained significant after adjustment for other antibodies, acute-phase reactant and conventional risk factors. Serological evidence of infection by multiple agents was also significantly associated with coronary events. Conclusions. Serological evidence for several infectious agents is associated with the risk of coronary heart disease, but only in men without baseline history of heart disease.

CCR5, a leukocyte chemoattractant receptor for chemokines CCL3, CCL4, and CCL5, promotes innate and adaptive immune responses by mediating leukocyte trafficking within lymph nodes and to peripheral tissues and is also known as a co-receptor for HIV cell entry. Homozygous inheritance of a complete loss-of-function mutation in CCR5 (CCR5Δ32/CCR5Δ32) is associated with symptomatic neuroinflammatory disease in humans with West Nile and Tickborne Encephalitis flavivirus infections. This study sought to establish whether CCR5 deficiency could also be a determinant of clinical outcome after infection by poliovirus which results in central nervous system damage in only a small proportion of cases. We analyzed serum samples from seven patients and 79 controls, collected during the 1984-1985 polio outbreak in Finland, where CCR5Δ32 is relatively common in the general population. The results excluded CCR5 deficiency as the sole determinant of severe neurologic disease after poliovirus infection in this population.

We carried out a randomized double-bind, placebo-controlled clinical trial on the topical treatment of recurrent mucocutaneous herpes with a strong water solution of Ascoxal®, an ascorbic acid-containing pharmaceutical formulation with mucolytic and non-specific antimicrobial activities. The lesion was firmly pressed with a cotton wool pad soaked in drug solution 3 times for 2 min with 30-min intervals on the first day only. Evaluation of the effects was by daily recordings of several different symptoms, including the presence and severity of erythema, induration, papulae or vesicles and scab by both the patient and a trained nurse, and by virus culture. Fourteen episodes with active treatment and 18 with the placebo were analyzed. According to the patients' records, the active treatment resulted in a significantly smaller cumulative number of days with scab (P < 0.01), or with any remaining symptom (P < 0.02) and significantly fewer occasions of worsening of any symptom after the treatment (P < 0.05). According to the nurse's records, the persistence of scabs was significantly shorter in the active treatment group (means 3.4 vs 5.9 days, P = 0.03). Virus culture after the first day of treatment yielded herpes simplex virus significantly less frequently in the active treatment group than in the placebo group (P < 0.01). In conclusion, a brief treatment with this ascorbic acid-containing preparation resulted in statistically significant clinical and antiviral effects, which calls for further and more extensive studies with a more intensive treatment schedule.

Some common viruses responsible for respiratory disease have been reported to be poor inducers of interferon (IFN). Therefore, we have studied the induction of IFN in cultures of human leukocytes exposed under standardized conditions to various concentrations of adenovirus type 7A, coronavirus 229E, an influenza type A virus (H3N2), a rhinovirus, and respiratory syncytial virus (RSV). All five viruses induced substantial amounts of IFN at a multiplicity of infection of one infectious unit per cell or less. Leukocyte cultures from 50 healthy children were exposed to a standard concentration of each of the viruses. IFN was induced almost without an exception, but the amounts produced varied extensively according to both the virus and the individual leukocyte donor.

Genetic defects in purine metabolism are associated with severe immunodeficiency. Adenosine deaminase deficiency impairs the function of both B- and T-lymphocytes whereas in purine nucleoside (inosine) phosphorylase deficiency there is more severe impairment of T-lymphocyte functions than of B-lymphocyte functions. The relative unimportance of the salvage pathway catalysed by hypoxanthine-guanine phosphoribosyltransferase is shown by the normal responses of T-lymphocytes from patients with the Lesch-Nyhan syndrome to antigenic and mitogenic stimulation. A mild deficiency of B-lymphocyte function is found in these patients. Agents inhibiting the de novo pathway of purine synthesis, including azaserine, 6-mercaptopurine and azathioprine in low doses, block the responses of normal human lymphocytes to mitogenic stimulation. These observations emphasize the importance of the de novo pathway of purine synthesis in lymphocyte responses to antigenic and mitogenic stimulation. There is considerable heterogeneity in the amount of labelled uridine incorporated into human and rat lymphocytes. This does not appear to reflect only a difference between T- and B-lymphocytes

The outbreak of paralytic poliomyelitis in Finland in 1984 was halted by nationwide oral poliovirus vaccination campaign. Immunocompromised patients, including those with chronic uraemia and on continuous dialysis, were excluded from the oral vaccination group and instead were given a dose of the new enhanced-potency inactivated poliovirus vaccine before the campaign. We studied the antibody response to this vaccine in 49 patients on chronic dialysis, using conventional antigen of all three serotypes and two additional type 3 strains. It was observed that 86% (42 of 49) of patients either had a satisfactory concentration of neutralising poliovirus antibodies against all three serotypes prior to vaccination, or responded with at least a four-fold increase of antibodies. Fourteen of 21 patients originally seronegative to at least one of the five virus strains used showed a striking seroconversion. One patient remained seronegative to type 1 poliovirus while two and four other patients were left with low (less than 8) titres of type 1 and 3 antibodies respectively. The latter seven patients showed moderate or good responses to the other two serotypes. We conclude that the enhanced-potency inactivated poliovirus vaccine produces a good antibody response in uraemic patients.

In 2005, 1983 cases of poliomyelitis were reported worldwide; 50 were caused by vaccine-derived polioviruses.

In 1988, the World Health Assembly established the goal of eradication of poliomyelitis by 2000. At the time, there were approximately 350 000 cases in 125 countries

Enteroviruses may be involved in the pathogenesis of Type 1 diabetes through different mechanisms including triggering of autoimmunity. The effect of immunization with coxsackievirus B4-E2 on diabetes incidence was studied in the non-obese diabetic mice, an animal model for human autoimmune insulin-dependent diabetes mellitus. The immunization delayed the onset of diabetes in the mice, and the effect was mediated at least partially by virus immunization-activated splenocytes as demonstrated by adoptive transfer experiments. Immunization resulted in a strong humoral immune response against the immunizing virus, formalin-inactivated coxsackievirus B4-E2. Cell-mediated immune response to virus antigen was characterised by interferon gamma and interleukin 10 secretion. The immunization also resulted in increased antibody levels against several beta-cell autoantigens. By using epitope mapping we were able to show that in addition to reactivity with the known epitopes of viral proteins and tyrosine phosphatase IA-2 or heat shock protein 60, responses to some other regions of autoantigens were enhanced. In preproinsulin, the response was restricted against an antigenic region earlier identified as DR4-dependent epitope. This reactivity can not be explained by homologous amino acid sequences and it is possible that enterovirus immunization might change the autoantigen specific TH1/TH2 balance in non-obese diabetic mice. In conclusion, our results suggest that coxsackievirus immunization increased humoral immune response to beta cell autoantigens and this was associated with a less destructive pathology for spontaneous diabetes in non-obese diabetic mice.

Sixty-eight laboratory strains representing 49 enterovirus, 10 adenovirus, and 3 reovirus serotypes were inoculated in a recombinant murine cell line expressing the human poliovirus receptor gene (L alpha cells). Only polioviruses caused cytopathic effect over a 10-day period. Likewise, only polioviruses were isolated, by use of L alpha cells, from 168 fecal specimens from children from developing countries. These results suggest that the recombinant L alpha cells can be used for selective isolation of poliovirus from clinical specimens.

Insulin-dependent (type 1) diabetes is characterized by progressive destruction of insulin-producing beta cells probably by autoreactive T lymphocytes. Viral infections, especially those caused by coxsackieviruses, are postulated to play a role in the pathogenesis of the disease in humans. One mechanism by which viral infections could initiate or accelerate diabetogenic processes is "molecular mimicry," induction of antiviral immune responses cross-reacting with epitopes in the beta-cell autoantigens. Tyrosine phosphatases (IA-2, IAR) represent a major target autoantigen in type 1 diabetes. Both humoral and cellular immune responses are directed to the carboxy-terminal (C-terminal) part of the protein. This region has a 5-amino acid sequence identity, followed by five amino acid similarity with the conservative motif in the VP1-protein of enteroviruses (PALTAVETGA/HT), which is a highly immunogenic B- and T-cell epitope in enterovirus infection-induced immune responses. This observation prompted us to investigate potential humoral cross-reactions between immune responses induced by tyrosine phosphatases and enteroviruses. The reactivities of various peptide- and virus-induced rabbit antisera clearly demonstrated that cross-reactions do exist, and in both directions. Using epitope mapping, we were able to show that several diabetes-linked epitopes in IA-2 were also recognized by CBV-4-induced antisera. Immunization of female NOD-mice with formalin-inactivated purified strain of coxsackievirus B4 (CBV-4-E2) induced an immune response that recognized the IA-2/IAR diabetogenic peptide. The results obtained with human paired sera, collected during enterovirus infection, indicated that enterovirus infection in humans may also occasionally induce a humoral response that cross-reacts with IA-2/IAR.

Surveillance for acute flaccid paralysis (AFP) in children younger than 15 years, and careful investigation of the cases, are the cornerstones for monitoring the progress of the poliomyelitis eradication programme. However, its sensitivity to detect wild type poliovirus (wtPV) circulation decreases when the incidence of true polio cases approaches zero. Under these conditions, only about one in 100,000 children is being investigated for poliovirus excretion. No real alternative approach which is generally exploitable has been developed. Environmental surveillance may in optimal conditions be at least as sensitive in detecting poliovirus circulation as AFP surveillance. However, optimal conditions, i.e. converging sewage systems, are not used by most people in the remaining endemic countries. Enterovirus surveillance, based on isolation of poliovirus in the routine diagnostic services, is only applicable in a few countries, where the diagnostic activity covers the entire population. Whichever approach is used, we will never reach 100% certainty of complete elimination of wtPV circulation. However, by applying all these approaches optimally, we may eventually reach a probability level allowing the safe cessation of immunisation.

In the recent years Echovirus-30 associated outbreaks have taken place in different European countries. Aseptic meningitis caused by Echovirus-30 was the main diagnosis of a large outbreak in Belarus in Summer-Autumn, 1997, involving 460 patients. Echovirus-30 was detected in cerebrospinal fluid of the patients with aseptic meningitis. This serotype played the dominant role in the outbreak. Minor serotypes and mixtures of enteroviruses were detected in faeces and nasopharyngeal lavages. Investigation of environmental samples gave evidence of expressed viral contamination of drinking water and water sources (river and ground sources). River water sources were considerably contaminated with viruses. The incidence of virus isolation was 50%. After cleaning procedures, the incidence became two times lower, proving imperfect water purification and disinfection procedures. Sequence analysis of isolates from Belarus (isolates from water and patient's cerebrospinal fluid) showed the difference of 0.2%. The outbreak peculiarities such as high attack rate and wide-spread of the disease incidences, clinical form variability, isolation of outbreak strain from water and a good agreement between minor serotypes isolated from faeces and water samples as well as correlation in the dynamics of acute intestine infections, aseptic meningitis morbidity and bacterial water contamination can be considered as evidence of its water-borne. Echovirus-30 isolates from Belarus were very closely related to each other and to several European isolates. Sequence difference between isolates of 1994-1998 from European countries was found to be 4.3%. The data can point to the common primary source of enterovirus infection, connected to water and to the possibility of epidemic strain transmission from neighbouring states to the Republic of Belarus.

In order to assess the feasibility of environmental poliovirus surveillance, known amounts of poliovirus type 1, strain Sabin, were flushed into the sewage network of Helsinki. Grab specimens collected at a remote downstream location and concentrated about a 100-fold revealed infectious poliovirus on four successive days in all three separate experiments. As for concentration, a simple two-phase separation method was found to be at least as useful as a several-fold more resource-demanding polyethylene glycol (PEG) precipitation method. Recovery of the introduced virus was remarkably high (more than 10%). Using the current system, it might be possible to detect poliovirus circulation in a population of 700,000 people by examining a single 400 ml sewage specimen, if 1 out of 10,000 inhabitants were excreting the virus. It is concluded that environmental surveillance is a sensitive approach to monitor silent poliovirus circulation in populations served by a sewage network.

In recent years outbreak of enterovirus infections caused by Echovirus-30 were rather frequently registered in different European countries. A major outbreak caused by this virus took place during the summer-autumn period of 1997 in the city of Gomel, Belarus. Sanitary epidemiological and molecular epidemiological studies made it possible to determine that the outbreak was water-borne. The sequence analysis of Echovirus-30 strains isolated from water and the cerebrospinal fluid of patients revealed a minor divergence between them (0.2%) indicative of their practical identity. The comparison of the Belorussian isolates with the strains isolated in Europe in 1994-1998 also showed a small percentage of differences in their genomes, which showed that the outbreak of Echovirus-30 infection was probably brought to Belarus from the territories of European countries.

Enteroviruses may be involved in the pathogenesis of insulin-dependent diabetes mellitus, either through direct beta-cell infection or as triggers of autoimmunity. In the present study we investigated the patterns of infection in adult human islet cell preparations (consisting of 56+/-14% beta-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells, but only CBVs caused cell death. One week after infection, the insulin response of the beta-cells to glucose or glucose plus theophylline was most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these cells. DNA fragmentation was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3% in controls; P0.01). CAV-9 infection did not induce DNA fragmentation. One week after infection the majority of infected cells showed characteristics of secondary necrosis. Medium nitrite and inducible nitric oxide synthase messenger ribonucleic acid levels were not significantly up-regulated by CBV infection. These results suggest that several enteroviruses may infect human beta-cells. The infection may result in functional impairment or death of the beta-cell or may have no apparent immediate adverse effects, as shown here for CAV-9. Coxsackie B viruses cause functional impairment and beta-cell death characterized by nuclear pyknosis. Apoptosis appears to play a minor role during a productive CBV infection in beta-cells.

In 1984 to 1985 an outbreak of poliomyelitis was found after a 20-year free period in Finland. The 10 patients with central nervous system disease were studied for HLA antigens. Additionally, two families with two and four affected siblings from the previous outbreak were analysed. The affection of the central nervous system seems not to depend on HLA-associated genetic factors.

In a prospective multicentre study described previously on prediabetic events in siblings of index cases with insulin-dependent diabetes mellitus, 31 children developed clinical diabetes during the observation period and 51 children seroconverted for islet cell antibodies or insulin autoantibodies. By using nonserotype specific EIA and RIA, it has shown recently that enterovirus infections in both groups were frequently associated with increases of islet cell antibody and/or insulin autoantibody titres. Serum specimens sequentially collected from 12 children during the prediabetic period were still available and were then tested for serotype-specific neutralizing antibodies. Plaque-neutralization assays were carried out for coxsackievirus A9, coxsackievirus B types 1 to 6, and echovirus types 1 and 11. An unequivocal monotypic increase in neutralizing antibodies was observed on seven occasions in six children, on one occasion with coxsackievirus A9, one with coxsackievirus B1, two with coxsackievirus B2, two with coxsackievirus B3, and one with coxsackievirus B5. In four patients, the infection was associated temporally with increases in the levels of islet cell antibodies, insulin autoantibodies and/or antibodies to glutamic acid decarboxylase, and in three other patients, it coincided with the clinical onset of insulin-dependent diabetes mellitus. These results suggest that the association of enterovirus infections with insulin-dependent diabetes mellitus is not restricted to serotype 4 of coxsackie B viruses suspected previously, but that several different serotypes might play a role in the pathogenesis of the disease.

Exposure to Coxsackie B virus or other enteroviruses prenatally or in childhood increases the risk for later manifestation of insulin-dependent diabetes mellitus (IDDM). The occurrence of enterovirus infections was analyzed in 23 initially nondiabetic and islet cell antibody (ICA)-negative siblings of IDDM patients who converted to ICA positivity during a prospective follow-up study. Increases in enterovirus antibody levels, documented by heavy chain-capture RIA and EIA techniques, were significantly more frequent in sample intervals in which ICA first appeared (18/23, 78%) than in other sample intervals in these siblings (30/92, 33%; P.001) or all sample intervals in 97 ICA-negative control siblings (117/403, 29%; P.001). The children who converted to ICA positivity during an enterovirus infection more often had the high-risk HLA-DQB1 genotype than did children who were constantly ICA-negative (P.01). The results suggest that enteroviruses may be important in the induction of a beta cell damaging process long before the clinical manifestation of IDDM.

This study aimed to test the hypothesis that viral and other microbial infections cause Bell palsy and to use the interferon (IFN) alpha/beta-induced MxA protein as an indicator of systemic viral infection.Bell palsy has been previously associated with several viral infections. Recently, after this study was completed, herpes simplex virus DNA was detected in the endoneurial fluid of some patients with Bell palsy.Serum and blood mononuclear cells were obtained from 30 patients with Bell palsy and 12 control subjects. The sera were tested for antibodies to 21 microbes. Mononuclear cells were assayed for (a) MxA protein using immunoblotting and (b) capacity to produce IFN in short-term culture after stimulation with influenza A virus.No significant differences were seen in serum antibodies or MxA protein between the patients and controls. The geometric mean of leukocyte IFN production in the convalescent phase of Bell palsy patients was higher than in the acute phase but remained still at significantly lower levels as compared with the control group (p0.05). In three patients there was no detectable IFN production.These results provide no evidence for a systemic viral involvement in Bell palsy, but the observed decreased IFN-producing capacity at the onset of Bell palsy could be a sign of transient immunosuppression or of an abnormal frequency of leukocyte subpopulations in the disease.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.

Rabbits were immunized with synthetic peptides derived from an immunodominant region of the VP1 capsid protein of enteroviruses. This region shows a high degree of homology among all sequenced members of the genus. Peptide-induced antisera were used for immunoperoxidase staining of cell cultures infected with 41 different serotypes of enterovirus. Specific cytoplasmic staining was readily seen in all but two cases. Echovirus type 22 was previously known to differ genetically from the rest of the enteroviruses, and hence, a negative result was expected. Surprisingly, one of the tested serum samples reacted with echovirus 22-infected cells. Coxsackievirus A7-infected cells could be reliably stained with only one of the tested serum samples. For the remaining 39 serotypes, scattered infected cells resulting from 1 to 2 days of incubation with diluted inocula were easily scored as positive before the cytopathic effect became visible. The same antibodies were also used in a sandwich-type enzyme immunoassay to demonstrate poliovirus antigens in cell extracts as early as 3 h after a high-multiplicity infection. These antibodies are candidates for enterovirus group reagents, being potentially useful in both the laboratory diagnosis of enterovirus infections and research on enterovirus-host interactions.

It has been known for some time that inactivated poliovirus vaccine (IPV), including the recently introduced enhanced-potency preparations, in spite of inducing very high levels of neutralizing and probably protecting serum antibodies are inferior to oral poliovirus vaccine (OPV) in preventing intestinal poliovirus infection in the vaccinees. We have proposed that this deficiency, at least as regards type 3 poliovirus, is based on different antigenic sites involved in the induction of antibodies by the two different vaccines because of proteolytic cleavage of a major antigenic site during infection. An approach to circumvent this problem comprises modification of the type 3 component of IPV with trypsin before immunization. A pilot vac cine prepared according to this principle has been obtained and its clinical evaluation will be started soon.

We have exposed 22 independent type 2 poliovirus isolates to human intestinal fluid and purified trypsin. In all cases the virus retained its infectivity, while polyacrylamide gel electrophoresis of viral proteins showed disappearance of the VP1 bands. Concomitantly, the viruses became resistant to antigenic site 1-specific monoclonal antibodies, indicating that the cleavage took place at the antigenic site 1. Sera from persons immunized solely with the inactivated poliovirus vaccine (IPV) neutralized intact type 2 polioviruses more readily than the corresponding trypsin-cleaved virus preparations. The ratio between the neutralization indices for the intact and trypsin-cleaved type 2 polioviruses was not significantly changed by a dose of trivalent oral poliovirus vaccine given to children previously immunized with IPV. These results indicate that while the antigenic site 1 of type 2 poliovirus is immunogenic in humans when IPV is used, the relative role of this antigenic site in human immunity appears to be less critical than that in the case of type 3 polioviruses. Before we obtained these results, only antigenic site 1 had been shown to be immunogenic in type 2 polioviruses.

Three patients with the Lesch-Nyhan syndrome were found to have normal delayed hypersensitivity, peripheral-blood T-lymphocyte counts, lymphocyte responses to P.H.A., and serum IgM, IgA, and IgE levels. However, the percentages of B-lymphocytes, IgG levels, serum-isohaemagglutinin titres, and lymphocyte responses to pokeweed mitogen (P.W.M.) were subnormal. These observations suggest that activity of the salvage pathway of purine synthesis catalysed by hypoxanthine-guanine phosphoribosyl transferase (H.G.P.R.T.) is not required for the responses of T-lymphocytes to mitogenic or antigenic stimulation, but may contribute to the proliferation and function of B lymphocytes. The major role of the de-novo pathway of purine synthesis in human lymphocyte responses to mitogenic or antigenic stimulation is shown by the effects of inhibitors of this pathway, including immunosuppressive agents, and by the effects of congenital deficiency or inhibition of adenosine deaminase.

Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol-bis-(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.

alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a radioimmunoassay. Unfractionated mononuclear leukocytes produced greater amounts of the macroglobulin (4.0 vs. 0.8 ng/10(6) cells) than did subpopulations enriched in T or B+T lymphocytes, by passage through nylon wool or cotton wool columns, respectively. Still higher concentrations of alpha2-macroglobulin (40 ng/10(6) cells) were measured in the supernates of glass-adherent mononuclear leukocyte cultures. These results suggest that cells of monocyte-macrophage lineage are mainly, if not exclusively, responsible for the appearance of alpha2- macroglobulin in the supernate of human peripheral blood leukocyte cultures. The de novo synthesis and release of alpha2-macroglobulin by cultured monocytes was demonstrated by immunoprecipitation of radioactivity from supernates of 32S-methionine-labeled glass-adherent cells. Antiserum against purified alpha2-macroglobulin was used in both Ouchterlony double diffusion and double antibody precipitation tests. SDS-polyacrylamide gel electrophoresis of immunoprecipitates showed that most of the radioactivity comigrated with authentic alpha2-macroglobulin subunit at about 160,000 daltons.

The absorption of erythromycin from two different enteric-coated preparations was evaluated in three groups of healthy volunteers. After a single dose, taken after an overnight fast, absorption was significantly better from enteric-coated pellets than from tablets; both the mean peak serum concentration and the peak mean level were higher (p less than 0.01) in all three groups, and the mean area under the serum concentration-time curve (AUC) was at least 65% larger. Eight out of 23 subjects showed no or only a very low serum concentration after the enteric-coated tablets. In a follow-up study, 250 mg doses were given 6-hourly for 3 days, and again the mean maximum serum concentration was significantly higher (p less than 0.05) after the pellets. In conclusion, enteric-coated pellets led to more regular and predictable absorption of erythromycin than did coated tablets.

THE Streptomyces antibiotic A23187, a divalent cation ionophore1, has been used as a tool in studies of the role of calcium in several cellular phenomena2–5. A23187 has a much higher affinity for divalent than monovalent cations, and the increased permeability induced by this ionophore in cellular membranes favours calcium and magnesium. X537A, another divalent cation ionophore6, also enhances fluxes of monovalent cations7 and forms lipid-soluble complexes with noradrenaline and some other organic compounds8. A23187 has often been referred to as a specific divalent cation ionophore although, to our knowledge, the evidence for the specificity is restricted to the lack of interaction with common monovalent cations like sodium and potassium1,2. In this paper we report experiments which show that A23187 is able to form lipid-soluble complexes with leucine and other amino acids at concentrations as low as or lower than those needed to form similar complexes with calcium.