Granulocytic Nuclei Research Articles

Uptake of pp65 in in vitro Generated pp65-Positive Polymorphonuclear Cells Mediated by Phagocytosis and Cell Fusion?

Objective: The cytomegalovirus (CMV) antigenemia consists of the detection of CMV pp65 in the nucleus of polymorphonuclear granulocytes (PMN), but it is unclear where and how PMN pick up virus particles or proteins. In an in vitro model for CMV antigenemia we investigated the mechanism of pp65 uptake by PMN that results in its expression in the nucleus. Methods: A series of inhibitors of different mechanisms was used to study the uptake of pp65 by PMN during coculture with CMV-infected endothelial cells and we performed a morphological analysis by light and transmission electron microscopy. Results: Nocodazole and cytochalasin B inhibited uptake of pp65 by PMN with 59.4 ± 14.1 and 73.3 ± 12.7%, respectively. The presence of anti-CMV hyperimmune globulin or lactoferrin during coculture reduced the number of pp65-positive PMN with 45.8 ± 7.0 or 40.6 ± 3.2%. Furthermore, a small number of the pp65-positive PMN obtained after coculture had fused to large cells with multilobed nuclei. PMN were observed that enclosed viral particles as well as free viral particles containing PMN in the cytoplasm. Conclusion: Fusion of viral particles with PMN and phagocytosis are both involved in the uptake of pp65.

Atypical antineutrophil cytoplasmic antibodies with perinuclear fluorescence in chronic inflammatory bowel diseases and hepatobiliary disorders colocalize with nuclear lamina proteins.

Antineutrophil cytoplasmic antibodies (ANCA) are frequently associated with chronic inflammatory bowel diseases (IBD) and hepatobiliary disorders. However, their target antigens have not been identified yet. Recently, we observed an atypical perinuclear ANCA fluorescence (p-ANCA) together with an intranuclear staining using ANCA-positive sera from patients with IBD and hepatobiliary disorders. This observation suggests that the target antigens are localized within the nucleus of neutrophilic granulocytes. To further investigate this hypothesis, we examined sera from patients with ulcerative colitis, primary sclerosing cholangitis, autoimmune hepatitis or systemic vasculitis on ethanol or formaldehyde-fixed neutrophils using confocal laser scanning microscopy and immunoelectron microscopy. Counterstaining with propidium iodide, a DNA-specific dye, showed that ANCA-positive sera in IBD and heptobiliary disorders react with intranuclear antigens at the nuclear periphery of the neutrophils. Double immunolabeling techniques revealed that nuclear lamina proteins, lamins A, C and B1, and lamin B receptor were colocalized with the antigen(s) recognized by atypical p-ANCA. No colocalization was observed with classical p-ANCA and antibodies against histones (H1-H4). Our study showed that atypical p-ANCA are antinuclear antibodies reactive with granulocyte-specific antigens present in the nuclear lamina.

Chromatin Structure in Granulocytes: A LINK BETWEEN TIGHT COMPACTION AND ACCUMULATION OF A HETEROCHROMATIN-ASSOCIATED PROTEIN (MENT)

To study the mechanism of heterochromatin formation in vertebrate cells, we isolated nuclei from chicken polymorphonuclear granulocytes and examined the chromatin organization. We found granulocyte chromatin to remain insoluble after nuclease digestion and to be resistant to swelling in low salt/high pH media. Both insolubility and resistance to swelling were lost after washing with 0.3 M NaCl, a procedure that released two abundant tissue-specific proteins from granulocyte nuclei. One of them (42 kDa) is identified as MENT, a protein previously shown to be associated with repressed chromatin from mature chicken erythrocytes. We show that MENT is immunolocalized in granulocyte heterochromatin, where it is one of the most abundant chromatin proteins ( approximately 2 molecules/200 base pairs of DNA). MENT is the first nuclear protein structurally related to the serine protease inhibitor family. The other abundant protein is similar to or identical with mim-1, a myeloid-specific protein that is known to be stored in cell granules and to associate with isolated nuclei. MENT (but not mim-1) binds chromatin and free DNA, and, at its physiological protein/DNA ratio, enhances compaction and the reversible Mg2+-dependent self-association of nucleosome arrays. MENT appears to promote the formation of heterochromatin by acting as a "glue" within and between chains of nucleosomes.

Mode of disease progression in primary myelodysplastic syndromes: A Japanese co-operative study

Chronological changes in hematological findings were analyzed in 225 patients with myelodysplastic syndromes (MDS). They were diagnosed between 1990 and 1992. Their hematological findings, i.e. hemoglobin levels, leukocyte and platelet counts, proportions of peripheral blood (PB) blasts and monocytes, and proportion of blasts in bone marrow (BM), were recorded for up to 42 months after diagnosis, when available. BM was examined regularly in only a few patients. Therefore, it was impractical to use the French-American-British Cooperative Group criteria for subtype classification during the disease course. Thus, we used the percentage of PB blasts as the only indicator of stage evolution. We classified the disease into four stages: stage 1, less than 1% PB blasts; stage 2, 1–5% PB blasts; stage 3, 5–30% PB blasts; and stage 4, 30% or more PB blasts. There were 171 patients initially in stage 1, 37 initially in stage 2, and 17 initially in stage 3. Less than half (45%) of the patients initially in stage 1 progressed to stage 2, while 91% of the patients initially in stage 2 and all of the patients initially in stage 3 showed stage evolution. Eight variables, i.e. BM blasts 5% or more, male sex, karyotypic abnormalities, micromegakaryocytes, mononuclear large megakaryocytes, platelet counts 50 × 10 9/l or higher, abnormal nucleus of granulocytes, and abnormal granules of granulocytes, were found to be significant risk factors for evolution from stage 1 to 2. Evolution from stage 1 to a higher stage within 15 months of diagnosis was associated with impending poor prognosis in most patients. However, of the 67 patients initially in stage 1 who died, 30 did not show stage evolution. Evolution from stage 2 to a higher stage and from stage 3 to stage 4 was also associated with impending poor prognosis. Higher levels of cytopenia were not associated with poorer prognosis in the stage 1 patients. In conclusion, our grading system proved to be useful in evaluating the chronological changes in MDS patients.

赤血球ＭＡＰ「日赤」に生成する巨大凝集塊（ＭＡ）について

The origin and formation mechanism of macroaggregates (MA) observed in RC-MAP, red cell preparation with additive solution MAP, were investigated here.MA were formed in all of RC-MAPS which were stored for 5 weeks at 4°C. In experiments of aggregates formation using the mixtures of each blood cell component and MAP solution, only the mixture of granulocytes and MAP solution formed experimental macroaggregates (exp-A) which was very similar to spontaneous MA in terms of appearance, elasticity and adhesiveness. Although both MA and exp-A were rapidly disintegrated by deoxyribonuclease I digestion, they were not digested at all by plasmin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions showed that most of proteins of MA were below 60kD and the band pattern was similar to that of granulocytes. In SDS-PAGE under reducing conditions of MA, fibrin bands were not detected.Leukocytes in RC-MAPs gradually decreased after 2 days of storage and the size distribution of leukocytes showed granulocytes to be decreased in number. On the other hand, platelets did not decreased until 14 days of storage. DNA contents of dried MA were constant, giving about 5.0%. The ratio of DNA of MA to total DNA in RC-MAP at 0 day of storage was inversely correlated with the ratio of leukocyte count in RC-MAP after separation of MA to 0 day of storage. But there was no relation between the DNA ratio and platelet count.Examinations by light microscopy and scanning electron microscopy showed that MA were composed of degraded leukocytes at large and variable numbers of leukocytes and erythrocytes, but no fibrin fibers.These results suggest that MA is formed by DNA fibers which are released from nuclei of granulocytes, taking up leukocyte debris and other blood cells. It does not seem that any coagulation factor or coagulation system is involved.

Raman microspectroscopic approach to the study of human granulocytes

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Nonhistone Protein Antigen Profiles of Five Leukemic Cell Lines Reflect the Extent of Myeloid Differentiation

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS-polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

Nonhistone protein antigen profiles of five leukemic cell lines reflect the extent of myeloid differentiation

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS- polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

Basophil Histamine Release Induced by Leukocyte Nuclei in Patients with Rheumatoid Arthritis

Leukocyte suspensions containing basophils were obtained from 23 patients with rheumatoid arthritis (RA). When these cells were incubated with leukocyte nuclei from normal persons, histamine release was seen in 11 of the 15 patients with active disease, but not in the quiescent group or in normal individuals. The dose-response curve for histamine release was similar to that obtained by specific antigen in type I allergy. By removal from and refixation to the cells of surface Ig, the release of histamine was respectively, abolished and restored, just as in similar experiments in hay fever patients. The dependence of pH for removal was also identical with that found in type I allergy. Antinuclear antibodies of the IgE class (IgE ANA) mainly directed against the granulocyte nuclei were often found in serum and on the cell surface of the RA patients, but not in normal individuals. A correlation was found between these titres in serum and on the cell surface. No correlation was found between ANA in serum and on the cell surface, on the one hand and disease activity and histamine release on the other. In a group of 12 patients with another joint disease, osteoarthrosis, only two patients showed histamine release, and in contrast to the other patients they showed swelling of more than two joints. The present investigation supports our hypothesis of an involvement of an autoimmune type I reaction directed against nuclear components in the RA disease.

Lactoferrin: nuclear localization in the human neutrophilic granulocyte?

Conditions were established whereby nuclear or cytoplasmic immunocytochemical localization of lactoferrin was observed in the human peripheral blood granulocyte. A positive nuclear staining reaction was obtained when cells were either not fixed or treated with most fixatives. However, treatment of blood smears with formaldehydeacetone prior to the immunocytochemical localization gave a cytoplasmic staining reaction. A method was developed that allowed us to examine proteins obtained from granulocyte nuclei isolated from fixed cells. Only a trace amount of lactoferrin was detected in the electrophoretically separated nuclear proteins obtained from granulocytes treated for 1 min with formaldehyde-acetone. However, lactoferrin was major protein component in nuclei isolated from untreated cells, cells treated with other fixatives, or cells preincubated in buffered saline prior to formaldehyde-acetone fixation. formaldehyde-acetone treatment of nuclear materials containing lactoferrin did not extract lactoferrin or mask it from detection, thus indicating that lactoferrin found in the mature neutrophilic granulocyte nucleus is most likely acquired from other cellular organelles during tissue processing.

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [ 3H]uridine and [ 3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3–4E cells with PE cells results in the incorporation of [ 3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.

The role of the nuclear protein matrix during development of rabbit gr anulocytes

A proteinaceous nuclear substructure (nuclear protein matrix or nuclear pore complex-lamina) has been described in a number of cells and may be a universal feature of cell nuclei. We have investigated the nuclear protein matrix (NPM) in the rabbit blood granulocyte and its precursor cells to determine (A) whether the NPM composition is similar to other cells that have been studied and (B) to determine whether the dramatic morphological changes that the granulocyte nucleus undergoes during cell maturation are related to changes in the composition or structure of the NPM. NPM preparations from rabbit granulocytes were similar but not identical to those found in HeLa cells or rat liver nuclei. The NPM structure of more mature cells retained more DNA during the isolation procedures than did immature cell NPMs, and the DNA was less accessible to DNAse in the mature cell nuclei. Scanning and transmission electron microscopy revealed a continuous outer covering of the NPM preparation and a lattice-like internal structure. Recognizable nuclear form persisted although the preparations represented less than 20% of the original nuclear protein. NPM preparations from mature cells were similar in overall dimensions and internal structure to NPM from immature cells, suggesting reexpansion during isolation of a previously compacted NPM structure in segmented neutrophils. NPM proteins are synthesized primarily in early stages of cell development. The NPM appears to play a major, but passive, structural role in the nuclear changes observed during maturation of granulocytes.

The Role of the Nuclear Protein Matrix During Development of Rabbit Granulocytes

AbstractA proteinaceous nuclear substructure (nuclear protein matrix or nuclear pore complex-lamina) has been described in a number of cells and may be a universal feature of cell nuclei. We have investigated the nuclear protein matrix (NPM) in the rabbit blood granulocyte and its precursor cells to determine (A) whether the NPM composition is similar to other cells that have been studied and (B) to determine whether the dramatic morphological changes that the granulocyte nucleus undergoes during cell maturation are related to changes in the composition or structure of the NPM. NPM preparations from rabbit granulocytes were similar but not identical to those found in HeLa cells or rat liver nuclei. The NPM structure of more mature cells retained more DNA during the isolation procedures than did immature cell NPMs, and the DNA was less accessible to DNAse in the mature cell nuclei. Scanning and transmission electron microscopy revealed a continuous outer covering of the NPM preparation and a lattice-like internal structure. Recognizable nuclear form persisted although the preparations represented less than 20% of the original nuclear protein. NPM preparations from mature cells were similar in overall dimensions and internal structure to NPM from immature cells, suggesting reexpansion during isolation of a previously compacted NPM structure in segmented neutrophils. NPM proteins are synthesized primarily in early stages of cell development. The NPM appears to play a major, but passive, structural role in the nuclear changes observed during maturation of granulocytes.

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.

Human granulocyte specific nuclear antigen(s). I: Production of antisera and determination of specificity

Non-histone protein-DNA complexes isolated from human lung tissue were used to immunize New Zealand rabbits. Quantitative microcomplement fixation and immunocytochemistry revealed a specific chromatin antigen(s) in the human granulocyte nucleus. The study demonstrates the feasibility of employing antisera against chromosomal non-histone protein-DNA complexes for the immunochemical identification of cell types.

Unmasking covert folate deficiency in iron--deficient subjects with neutrophil hypersegmentation: dU suppression tests on lymphocytes and bone marrow.

Fifteen patients with apparently uncomplicated iron‐deficiency anaemia were studied to determine the presence of covert folate or vitamin B12 deficiency. None had morphologic or conventional biochemical evidence of folate or vitamin B12 deficiency on admission, except for neutrophil hypersegmentation in peripheral blood smears of 10 of the 15 patients, and giant metamyelocytes in some of the bone marrow aspirates.Although deoxyuridine (dU) suppression tests on pretreatment bone marrow were normal in all 15 patients, similar tests on pretreatment transformed lymphocytes were characteristic of folate deficiency in eight of 14 patients (57%), with poor transformation obscuring results in the fifteenth subject.After six patients were given 6 weeks of oral folate and iron therapy, the dU suppression test was repeated in the lymphocytes of two subjects who demonstrated folate deficiency in this test on admission. The test still showed folate deficiency, supporting our prior report that the test was useful as a marker of past folate deficiency despite intervening folate therapy.The nine remaining subjects received iron alone for 3–6 weeks, which produced no change in serum folate, red cell folate, or dU suppression tests in lymphocytes. In five of the nine subjects (56%), however, an abnormality characteristic of folate deficiency appeared for the first time in bone marrow dU suppression tests, with morphologic megaloblastosis developing frankly in four and equivocally in one. Four of these five subjects had a folate corrected defect in the lymphocyte dU suppression test before treatment, and the fifth was the subject in whom lymphocytes did not transform sufficiently to allow testing for the defect.These observations: 1. Suggest that when a patient with apparently uncomplicated iron deficiency has hypersegmentation of his granulocyte nuclei, underlying folate deficiency should be sought. It was found in nine of the 10 current such subjects. 2. Suggest that the most sensitive test to detect covert folate deficiency in contrary to the situation in severe uncomplicated deficiency of vitamin B12 or folate, in which hypersegmentation is striking and rapid scanning of a hundred neutrophils yields a minimum of five with more than five lobes.In uncomplicated iron deficiency, iron binding capacity is almost always elevated above 400 μg/dl. Such levels were seen in only four of our 15 patients, two without hypersegmentation and two with hypersegmentation. This may suggest that a patient population with relatively low total iron binding capacity, perhaps often in association with liver disease, is more likely to have covert folate deficiency with their iron deficiency.

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[ U- 14C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.

Low serum vitamin B12 levels in patients receiving ascorbic acid in megadoses: studies concerning the effect of ascorbate on radioisotope vitamin B12 assay2–3

Serum vitamin B12 levels were found to be low in four of 18 hospitalized patients with traumatic spinal cord injury, who had been receiving 2 g daily of oral ascorbic acid as a urine acidifier for varying periods of time. Bone marrow studies, on two of the patients with low B12 levels, showed normoblastic erythropoiesis and a normal deoxyuridine suppression test. One of the two, however, had hypersegmentation of the granulocyte nuclei. Three control subjects showed no change in assayable B12 levels after 2 weeks of such ascorbate supplementation. In vitro studies demonstrated that in the absence of added KCN the addition of sodium ascorbate to normal serum, liver homogenate, or crystalline B12 solution before, but not after, the samples were heated reduced the amount of radioassayable B12. This study demonstrates that serum B12 determinations performed without added KCN may be low due to the presence of ascorbate in the serum, and that such low levels do not necessarily reflect tissue deficiency of the vitamin. Therefore, to adequately interpret a low serum B12 level, it is important to know if the patient has been taking large doses (in the range of 0.5 g four times daily) of ascorbic acid, by prescription or as self-medication. In situations where neither heat alone nor ascorbate alone will destroy B12, both together will unless the B12 is protected against such destruction by conversion to a stable form, such as cyanocobalamin. We now add cyanide before the heating step in B12 radioassay, to prevent ascorbate destruction of vitamin B12, unless the assay is carried out at pH 9.3.

Cryoimmunoglobulinemia in Felty's syndrome.

Seven of 9 patients with Felty's syndrome exhibited cryoglobulins in greater amounts than found in uncomplicated RA but similar to amounts found in RA with vasculitis. Antinuclear antibodies were found in high titer in the sera and cryoglobulins of Felty's patients; when compared to SLE patients, whose sera and cryoglobulins also contained antinuclear factors, an antinuclear antibody reacting with nuclei of polymorphonuclear granulocytes appeared to be selectively concentrated in the cryoglobulins of some Felty's patients.

RECIPROCAL CHANGE WITH AGE IN ANTIBODY TO EXTRINSIC AND INTRINSIC ANTIGENS

A reciprocal relation was found with age between "natural" antibody to an "extrinsic " antigen, flagellin, and antibody to an " intrinsic " (auto)antigen, the granulocyte nucleus. Sera were tested from 400 healthy subjects aged 20-89 years grouped by decades of age and paired for sex. The serum of 85% gave a tanned-red-cell-hæmagglutinin reaction with flagellin from Salmonella adelaide, despite no known exposure to this organism. This "natural" antibody was predominantly IgM, titres were higher in young women than young men, and mean titres were significantly higher in younger than in older decades. By contrast, the incidence of positive reactions and mean titres for antinuclear antibody increased with age, more so in women. These findings seem relevant to ageing, particularly decreased resistance to infections and increased predispositon to autoimmune reactions with age.