Recombination at his-3 in Neurospora declines exponentially with distance from the initiator, cog.

By deletion of 1.8 kb of sequence between cog(L) and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog(L) and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog(L), a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His(+) recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log(10) (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog(L) in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.

Human ovary and placenta express messenger RNA for multiple activin receptors.

In the present study, we examined the expression of activin receptor (ActR) mRNAs in human ovary and placenta. Primers specific for two type I and two type II activin receptors (ActR-I, ActR-IB, ActR-II, and ActR-IIB) were used in polymerase chain reaction (PCR) to amplify cDNAs prepared from granulosa-luteal cells, placental tissues and isolated trophoblast cells. PCR products with the expected sizes for ActR-I, ActR-IB, ActR-II, and ActR-IIB mRNAs were detected in freshly dissociated and 5-day cultured granulosa-luteal cells; and in trophoblast cells from both first trimester and term placentas. The identity of these PCR products were confirmed by Southern blot hybridization, as well as cloning and sequencing. These results suggest that multiple activin receptors are present in human ovary and placenta and may mediate activin function in these tissues. The demonstration of activin receptor mRNAs in granulosa-luteal and trophoblast cells further supports the notion that activin is an important local regulator in the human ovary and placenta.

Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL2 receptor p55 chain (TAC, CD25).

Using a high-sensitivity immunofluorescence procedure, a proportion of blood lymphocytes can be shown to express the p55 chain of the IL-2 receptor (CD25, TAC) without in vitro stimulation. In this study, we measured the proportion of peripheral blood lymphocytes expressing CD25 in patients with a diagnosis of HIV infection and compared the results with cells from controls. The mean value for HIV-positive samples was 15%, while controls gave a mean value of 31%. The difference between the groups was highly significant (P less than 0.001, Mann-Whitney U test). When expression of CD25 was examined separately in CD4-positive and CD8-positive T cells and in B cells, there was a wider spread of values in the HIV group compared with controls, but no systematic difference. In the patient group studied (110 samples from 53 patients) there was a weak correlation between CD25 and CD4 expression (correlation coefficient r = 0.21, P less than 0.02), but there were patients with low CD25 and high CD4 as well as patients with high CD25 and low CD4, suggesting that CD25 can vary independently of changes in CD4 lymphocytes. Although the majority of very low values of CD25 (less than 10%) were found in patients with stage IV disease, such low values were also common in Stage II disease.

Expression of the p70 chain of the IL-2 receptor on human lymphoid cells: analysis using a monoclonal antibody and high-sensitivity immunofluorescence.

Using monoclonal antibody (MoAb) against the p70 (beta) chain of the interleukin-2 receptor (IL-2R) and a high-sensitivity immunofluorescence procedure, p70 can be demonstrated on the majority of lymphocytes from normal blood samples, without in vitro stimulation. A subpopulation of cells bears a high concentration of receptor, and these cells have the physical properties of large granular leucocytes (LGL). The smaller lymphocytes, although weaker, are quite clearly positive. T8 cells stain relatively strongly, while T4 cells and B cells stain relatively weakly. Leukaemic cells showed a variety of phenotypes when examined for expression of the p55 and p70 chains of the IL-2R. The demonstration of IL-2R chains directly by immunofluorescence on unstimulated lymphocytes has potential clinical applications, since this technique can be used in a diagnostic setting.

Low molecular weight IgM in juvenile chronic arthritis.

Low molecular weight IgM, the monomeric subunit of pentameric IgM, was clearly detected by immunoblotting and filtration chromatographic techniques in six patients with juvenile chronic arthritis and in trace quantities in a further eight of 24 patients studied. This low molecular weight IgM moiety contributed up to 33% of the total circulating IgM and was strongly associated with raised serum concentrations of IgM and the presence of antinuclear antibodies, extractable antinuclear antibodies, and rheumatoid factor. Immunoblot analysis of positive serum samples showed small quantities of other low molecular weight oligomers of IgM in addition to monomeric IgM. It is postulated that the presence of low molecular weight IgM in the serum of patients with juvenile chronic arthritis reflects a disorder of the intracellular assembly of IgM subunits during a stimulated IgM immune response. The pathogenetic role of low molecular weight IgM remains uncertain.

Large quantities of low molecular weight IgM in mixed cryoglobulinaemia.

Low molecular weight (LMW) IgM is the monomeric subunit of pentameric IgM. It was not found in the sera of 20 healthy subjects but was detected in all six patients with mixed cryoglobulinaemia with a mean value of 1.4 g/l, representing 34% of the total IgM. In three of four patients studied LMW IgM was monoclonal and of the same light chain type (kappa) as the pentameric monoclonal IgM rheumatoid factor (RF) observed in the cryoprecipitate. LMW IgM was proportionately under-represented, however, in the cryoprecipitate compared with the corresponding serum, possibly because of the lower valency of the LMW molecule. Immunoblot analysis of sera showed the presence of other oligomers of IgM in addition to monomeric IgM, suggesting that a disorder of IgM assembly was responsible for its occurrence, and this was supported by the secretion of large proportions of LMW IgM in vitro by peripheral blood mononuclear cells (PBMC) from one patient with this disorder but not from healthy controls. In conclusion, the occurrence of large quantities of monoclonal LMW IgM in mixed cryoglobulinaemia was observed, and it is suggested that this is unlikely to have a direct pathogenic significance. It is postulated that its presence reflects a disturbance of assembly of the monomeric IgM subunits that occurs during the polymerization of the pentameric molecule.

Frequency of low molecular weight IgM in human cord blood.

Thirty cord blood sera from healthy neonates and five sera from still-born infants (two with suspected infections and high IgM) were assessed for the presence of low molecular weight (LMW) IgM using two independent sensitive techniques, viz. filtration chromatography and immunoblotting. The first technique revealed this LMW moiety in 4 of 22 sera, all from healthy full-term infants, and it constituted 4-25% of the total IgM. LMW IgM was not found in any of the 30 sera using the immunoblotting technique or in 15 healthy adult sera, but was found consistently in rheumatoid sera used as positive controls.

Serological investigations in the diagnosis and management of infective endocarditis.

In a retrospective study of 39 patients with infective endocarditis (IE) all had elevated concentrations of C reactive protein (CRP) at presentation, patients with the acute variety having significantly higher values than patients with the subacute variety. In addition, the majority of patients with subacute bacterial endocarditis had elevated concentrations of circulating immune complexes (CICs) and rheumatoid factor (RF), both of which were absent in all but one of nine patients with acute bacterial endocarditis. Two patients with subacute and one with acute bacterial endocarditis had low values of C3 and C4. Measurement of CRP, CICs, and RF did not distinguish between patients with and without extracardiac manifestations. Sequential analysis of patients revealed that a successful response to antimicrobial treatment was indicated by a striking and rapid decline in CRP, with less striking declines in CICs, RF, and IgM. Antibiotic failure was indicated by the persistence of high concentrations of CRP and CICs. We conclude that the measurement of C reactive protein is of some value in the diagnosis and management of infective endocarditis. A normal CRP concentration excludes this diagnosis. The measurement of CRP alone appears sufficient for monitoring most cases of infective endocarditis with the sequential measurement of rheumatoid factor and circulating immune complexes adding no useful information except where the CRP remains elevated despite treatment. In this latter instance, persisting high levels of CRP and circulating immune complexes together herald an ominous course.

Appearance of low molecular weight IgM during course of infective endocarditis.

Low molecular weight (LMW) IgM is the monomeric subunit of pentameric IgM. It is not found in healthy adults but occurs in a number of autoimmune, lymphoproliferative and infectious conditions. It has not been described before in infective endocarditis (IE). Eighteen patients with IE were studied; 16 with subacute bacterial endocarditis (SBE) and two with acute endocarditis. LMW IgM was detected in the sera of six patients, all having SBE in association with circulating rheumatoid factor (RF). Of the remaining 12 patients without LMW IgM only three had RF in low quantities. Sequential studies revealed that LMW IgM appeared during the later stages of the illness at or following the peak RF and IgM response. LMW IgM was not detected in any of 20 control sera. Immunoblot analysis of sera containing LMW IgM revealed the presence of small quantities of dimeric and oligomeric IgM in addition to monomeric IgM. We conclude that LMW IgM occurs predominantly in those patients with IE who have associated RF. Immunoblot analysis suggests that the presence of monomeric and oligomeric LMW IgM reflects a disorder of IgM polymerization occurring in those patients.

Low molecular weight IgM in B cell lymphoproliferative disorders.

Circulating low molecular weight (LMW) IgM was demonstrated in five of 38 patients with B cell lymphoproliferative disorders. These five patients all had malignant disease and could be subdivided into two groups. In the first group were three patients, each with an associated serum IgM paraprotein; two had Waldenström's macroglobulinemia, and one lymphocytic lymphoma. The two patients of the second group did not have IgM paraproteins; one had lymphocytic lymphoma and one chronic lymphocytic leukemia. Both these patients also had acquired C1 esterase inhibitor deficiency, a previously recognised association with circulating LMW IgM. None of the 16 patients with benign IgM macroglobulinemia had circulating LMW IgM. In those positive sera with LMW IgM this moiety contributed between 10.5% and 37.5% of the total IgM. There was no apparent association between LMW IgM and total IgM levels, kappa/lambda typing or the presence of Bence Jones proteinuria, but rheumatoid factor, immune complexes and cryoglobulins occurred in many of the sera which contained LMW IgM. Pokeweed mitogen stimulated peripheral blood mononuclear cells from two patients with circulating LMW IgM secreted considerable quantities of this moiety in vitro but this did not occur in two patients with benign IgM macroglobulinemia. We conclude that LMW IgM is found in the malignant but not the benign forms of B cell lymphoproliferative disorders and is frequently associated with other serological abnormalities. The basic abnormality causing defective IgM polymerisation in these disorders is obscure.

Competitive inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM.

Competitive-inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM are described. These assays can be readily performed with commercial antisera and a recently developed method for purifying human IgA and IgM with high yield. The assays described are specific, with undetectable (less than 0.5%) cross-reactivity between the immunoglobulin classes in all systems, except with purified IgM, which cross-reacted to 1.9% with the IgG enzyme immunoassay. Minimal detectable concentrations of 2.5 +/- 0.8 ng/ml for IgG, 4.2 +/- 0.9 ng/ml for IgA and 7.2 +/- 1.4 ng/ml for IgM were recorded, indicating that these assays are particularly sensitive. There is little within-assay variation (mean coefficient of variation = 3.9%), although the between-assay variation was substantially greater (mean coefficient of variation = 23.5%). These assay systems appear to be particularly suited to the measurement of immunoglobulin production by lymphocytes in culture. In such studies the assay must be specific, sensitive and be capable of discriminating between levels of immunoglobulin produced in response to various experimental treatments.

The rosette inhibition test in early pregnancy diagnosis.

The rosette inhibition test (RIT) was used to detect pregnancy around the time of implantation. Using an antiserum to peripheral blood lymphocytes, it was possible to diagnose 80 per cent of pregnancies. These results substantiate the view that there is an early pregnancy factor (EPF) present in the serum at a very early stage of gestation.

PIP: The rosette inhibition test (RIT) was assessed for its ability to diagnose pregnancy around the time of implantation. A blind study was conducted on 20 sera consisting of 10 samples from pregnant women and 10 samples from nonpregnant women and males. Sera from pregnant women were collected 7 days postovulation from 10 women who attended the Flinders Medical Centre (Bedford, Australia) Fertility Clinic for artificial insemination with donor sperm and who subsequently were shown to have had a conceptual cycle. In the nonpregnant group, 6 of the samples wre from males and 4 were from women who were inseminated but who did not conceive during the treatment cycle and subsequently expierenced normal menstruation. Blood was collected from a male donor into sodium heparin tubes and the lymphocytes isolated using the Ficol-Hypaque method. 3 New Zealand white rabbits were injected weekly with 1 ml of cell suspension intravenously (into the ear vein) for 5 weeks. The rabbits were bled for antilymphocyte serum (ALS) 1 week after the 4th and 5th injections and the sera separated and stored at -70 degrees. The ALS was inactivated at 56 degrees Centigrade for 30 minutes before use. 6 male guinea pigs were bled monthly for complement. The RIT was carried out using the technique of Morton et al. (1977) with modifications. The rosette inhibition titre was taken to be reciprocal of the highest diluation of ALs in which there was 25% inhibition (or greater) of rosette formation compared to that observed in the control. 8 of the 10 samples from pregnant women had titres of 8 x 100,000 and the remaining 2 had tires of less than 4 x 100,000. It was possible to confirm 8 very early pregnancies in the 10 sera from women subsequently proved to be clinically pregnant. In the nonpregnant group, 1 serum sample from a female who exhibited no clinical evidence of pregnancy gave a titre of more than 8 x 100,000. Like Morton et al. (1977, the presence of an early pregnancy factor (EPF) was detected in the sera of a high proportion of women at a very early stage of pregnancy. The sera were obtained 7 days after ovulation and therefore presumptively around the time of implantation. The incidence of false positiveresults for the diagnosis of very early pregnancy appears to be quite low with this assay.

Disappearance of IgG2B autoantibodies associated with recovery from anaemia.

Mice immunized with cross-reacting rat erythrocytes develop autoimmune haemolytic anaemia as indicated by haematological data and erythrocyte autoantibodies. With continued immunization the mice recover haematologically but remain Coombs' positive. Coombs' tests were performed using monospecific antisera to determine whether the recovery from anaemia was associated with a change in the class or subclass of the autoantibodies produced. In both splenectomized mice and in unsplenectomized mice the following subclass of erythrocyte autoantibodies were present: IgG1, IgG2A, IgG2B. IgA autoantibodies were not detected and IgM autoantibodies were only detected in splenectomized mice 1-3 weeks after the IgG autoantibodies had appeared. After six immunizations the frequency of IgG2B autoantibodies decreased and by the tenth immunization and thereon, IgG2B autoantibodies were not detected. It is proposed from these results that the anaemia is caused by IgG2B autoantibodies and the the sudden exacerbation in the anaemia that occurs in splenectomized mice is due to the production of IgM autoantibodies.