A quantitative method for measuring in vitro synthesis of IgA and IgG by human rectal mucosa: studies on normal controls and patients with hypogammaglobulinaemia.

A quantitative technique has been developed for measurement of immunoglobulin production by human rectal mucosa in vitro. The technique overcomes the problem of serum proteins trapped in the tissue by parallel measurements of Ig and human serum albumin (HSA) over a 6 day period. IgG, IgA, IgM and HSA were measured in supernatant fluids using sensitive radioimmunoassays. The technique has demonstrated IgG and IgA synthesis by rectal mucosa in vitro. The conclusion that the observed IgA and IgG was synthesized in vitro was supported by the demonstration that the production could be increased by pokeweed mitogen and blocked by emetine. This culture system has been applied to measure Ig synthesis by the rectal mucosa of immunodeficient patients.     

Antiinflammatory effects of endotoxin. Inhibition of rabbit polymorphonuclear leukocyte responses to complement (C5)-derived peptides in vivo and in vitro.

Although capable of provoking a variety of inflammatory effects, endotoxin (bacterial lipopolysaccharide) paradoxically has been reported to be antiinflammatory. The authors have found that single intravenous injections of Escherichia coli endotoxin, 24 hours before challenge, inhibit almost completely the vascular permeability changes and exudation of polymorphonuclear leukocytes induced in rabbit skin by reversed passive Arthus reactions. Whereas intravenous injections of endotoxin also caused modest inhibition of the vascular permeability changes induced in rabbit skin by the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), exudation of polymorphonuclear leukocytes was unaffected. Polymorphonuclear leukocytes from rabbits given single injected doses of endotoxin exhibited markedly diminished chemotactic and degranulation responses to complement (C5)-derived peptides in vitro. Responses of these cells to FMLP, however, were normal. These data suggest that selective suppression of polymorphonuclear leukocyte responses to C5-derived peptides accounts, in part, for the antiinflammatory effects of endotoxin.     

Antigenicity of influenza virus hemagglutinin following chemical modification

Hemagglutinin (HA) molecules from a number of different strains of type A influenza virus were reacted with 1-fluoro 2–4-dinitrobenzene (FDNB) at pH values ranging from 8.4 to 10.3. HA was also reacted with tetranitromethane (TNM) or diazotized sulfanilic acid (DSA). Sequence studies on HA1 from HA molecules treated with FDNB, TNM, or DSA showed that certain lysine, tyrosine, or histidine residues were 100% substituted after the reaction, while others apparently did not react at all. DNP-substituted hemagglutinin molecules, isolated from FDNB-treated A/Memphis/1/71_H-BEL_N(H3N1) virus particles, had up to 58% of lysines substituted with DNP. These molecules, nevertheless, retained hemagglutinin activity and, as far as could be measured, the same capacity as the unsubstituted hemagglutinin to react with heterogeneous antiserum or a panel of monoclonal antibodies. These results suggest that those amino acid side chains able to react with FDNB (lysine, histidine, tyrosine, and cysteine) are either not present in the antigenic sites on the HA, or if they are, then either the side chains which bind antibody do not react with DNP, or the presence of DNP in the site does not affect its ability to combine with antibody. The results also suggest that substitution of more than half of the lysine in the hemagglutinin molecule does not cause any marked conformational changes, for such changes would be expected to affect the ability of the HA to combine with both cell receptors and antibody molecules. Similar findings with TNM-treated HA suggest that tyrosine is not an essential part of any antigenic site on H3 type HA. HA treated with diazotized sulfanilic acid lost HA activity, but its antigenicity was similar to that of untreated HA when tested with heterogeneous antisera, suggesting that histidine was not present in the antigenic sites. However, when HA from a monoclonal variant of A/Mem/1/71 (H3N2) virus with a sequence change from wild-type in HA1 of proline (143) to histidine (Laver, Air, and Webster, 1981) was reacted with diazotized sulfanilic acid, the histidine at position 143 in HA1 reacted completely and the HA lost the ability to bind antibody specific for the new antigenic site on this variant. However, treatment of this variant with FDNB did not lead to substitution of histidine 143.     

Retinoblastoma and p53 gene product expression in breast carcinoma: Immunohistochemical analysis and clinicopathologic correlation

We examined 100 breast cancers for retinoblastoma (Rb) and p53 protein expression by immunohistochemistry using the PMG3.245 and PAb 1801 antibodies. We assessed percentages of reactive cells and their intensity, as well as staining patterns. The results were correlated with neu protein reactivity and a panel of variables, including age, tumor size and type, nuclear grade, estrogen receptor/progesterone receptor content, and lymph node status. Retinoblastoma protein negativity, either partial or complete, was noted in 47% of cases. Surprisingly, a relatively stronger Rb reaction was seen in some high nuclear grade tumors. p53 positivity was found in 23% of cases and was a significant predictor of Rb loss. p53 also was correlated with poorly differentiated (nuclear grade III) neoplasms and neu expression but not with negative ER status. Tissue distribution profiles for Rb-negative and p53-positive cells were variable in this series, with both uniform and heterogeneous patterns observed. This suggests that Rb and p53 alterations may represent early or late events in transformation. Our findings further implicate Rb and p53 derangements in mammary oncogenesis.     

Characterization of antibodies to sporozoites in Plasmodium falciparum malaria and correlation with protection.

The antibody response to sporozoites of Plasmodium falciparum and the role of these antibodies in protection against malaria have not been systematically investigated. An understanding of antisporozoite antibodies in natural infection is, however, important to the development of a human malaria vaccine. In a prospective study in Thailand, an antibody response to sporozoites was observed only in individuals who developed parasitemia. Antibodies were detected against an epitope in the repeat region of the circumsporozoite (CS) protein. Current candidate sporozoite vaccines are based on CS repeat antigens. The CS antibody response was of low magnitude, peaked after detection of parasitemia, and had a serum half-life of less than 1 month. CS antibody boosting occurred in only 6% of reinfected individuals. These observations suggest that antisporozoite antibody is poorly developed under natural conditions and appears not to protect against development of malaria.     

Iso-frequency 2-DG contours in the inferior colliculus of the awake monkey

Summary Tone bursts produced bands of selective 2-[¹⁴C]-deoxyglucose labelling in the inferior colliculus (IC) of the awake monkey. Low tone frequencies produced labelling in dorsal regions and high tone frequencies produced labelling in ventral regions. The position of the bands coincided with the position of a single unit with a characteristic frequency, which was the same as the frequency producing the labelling. These findings indicate that the bands of labelling represent iso-frequency contours in IC. The iso-frequency contours extended across most of the nucleus and were oriented from dorsomedially to ventro-laterally at 20–30° from the horizontal and became more vertical anteriorly. The width of the contours was as narrow as 200 m, suggesting that the contours might represent 2 or 3 overlapping cellular laminae.Supported by research grants from the National Health and Medical Research Council of Australia and the Australian Research Grants Scheme     

Antigenic and structural characterization of multiple subpopulations of H3N2 influenza virus from an individual

Influenza viruses grown in embryonated chicken eggs frequently possess antigenically distinguishable hemagglutinin (HA) compared to virus from the same source grown in mammalian cell culture. To further investigate the extent of variation among viruses from an individual, viruses were isolated from throat washes collected over a 48-hr period during infection with influenza virus designated A/Mem/6/86 (H3N2). Viruses were isolated from limit dilutions in eggs and mammalian Madin-Darby canine kidney (MDCK) cells and the antigenic, structural, and receptor-binding properties of these viruses were determined. Viruses which could be isolated in MDCK cells were present at 10- to 100-fold higher frequency in the original sample than viruses which could be isolated in eggs. The HA of virus clones isolated in MDCK cells were antigenically and structurally identical. In contrast, viruses from the same source, selected at limit dilution in eggs, could be divided into three distinct subpopulations based on the distinguishable antigenic and structural characteristics of their HA molecules. The three groups of egg-grown viruses could be distinguished from each other, and from MDCK cell-grown viruses, not only by a panel of anti-HA monoclonal antibodies, but also by immune ferret sera raised to H3N2 virus strains of recent years and sera raised to the different egg-grown clones themselves. Of these groups, group 1 and group 2 egg-grown viruses each represented a minor subpopulation of viruses which could be isolated in eggs, while viruses of the third antigenic phenotype were the most frequently isolated in eggs. Amino acid substitutions in the HA of egg-grown viruses occurred in antigenic and receptor-binding sites of the molecule. Group 1 viruses each possessed two amino acid substitutions in their HA molecules at residues 193 and 229 in HA1. Group 3 viruses, which displayed altered receptor specificities compared to MDCK cell-grown viruses and other egg-grown viruses, possessed a single amino acid substitution at residue 145 in HA1. The HA of the group 2 egg-grown viruses appeared structurally identical, yet displayed marked differences in antigenic and receptor-binding properties, compared to viruses isolated in MDCK cells. These results demonstrate that multiple, distinct subpopulations of virus can be isolated from a single patient during an infection with influenza and highlights the potential problems in selecting the most appropriate virus for epidemiological and vaccine purposes since selection could result in the use of viruses that are not representative of those which predominate in a human population.     

Sequence analysis of cDNAs for the human and bovine ATP synthase β subunit: mitochondrial DNA genes sustain seventeen times more mutations

We have cloned and sequenced human and bovine cDNAs for the subunit of the ATP synthase (ATP-synß), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsynß was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsynß proved to be 1.9 × 10^–9 substitutions per site per year (substitutions × site^–1 × year^–1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the selective constraint ratio. This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.     

LFA-1-dependent OKT3-driven T cell clusters in common variable immunodeficiency.

The triggering of the TCR/CD3 complex by anti-CD3 (OKT3) antibody leads to the formation of T cell clusters. In cultures of T lymphocytes from most normal individuals, the peak of cluster formation occurs at 24 h, but with cells from patients with common variable immunodeficiency (CVI) it was seen earlier at 4-9 h; in addition, the clusters were larger than normal, particularly at 9 h. Cluster formation by CVI and normal cells was dependent on temperature and divalent cations, but did not require Fc receptors. Since OKT3 clustering is known to be dependent on the LFA-1/ICAM-1 adhesion system, the effect of monoclonal antibodies directed against these molecules was tested. A potent inhibitor was the antibody against the common beta chain of the integrin family (CD18), but of four MoAbs against the alpha chains (CD11), three inhibited and one stimulated T cell aggregate formation. Increased expression of LFA-1 or ICAM-1 on CVI patients' T cells could not be demonstrated. The accelerated clustering was therefore probably due to an increase in the proportion of cells carrying the activated form of LFA-1. The formation of large numbers of homotypic lymphocyte clusters might reduce the effective interaction between B and T cells, thus contributing to the depression of immunoglobulin synthesis observed in this disease.