Subfractionation of human peripheral blood lymphocytes by partitioning in two-polymer aqueous phase systems. Characteristics of a small subpopulation of cells with high partition coefficient

Mixtures of aqueous solutions of dextran and poly(ethylene glycol) form immiscible 2-phase systems that are suitable for the separation of viable, functionally-competent cells on the basis of their surface properties. With the appropriate choice of ionic composition, these systems will separate cells on the basis of charge-related surface characteristics. We have previously shown that in charge-sensitive phase systems, human B lymphocytes have a low partition coefficient (K) and T cells have an intermediate K. The cell subpopulation with the highest K values contains large null lymphocytes that have most of the antibody-dependent and natural killer activities. The present study further characterizes this latter cell subpopulation. 8-33% of peripheral blood lymphocytes in normal persons had high K values. About 50% of these cells had typical Giemsa-staining granules characteristic of killer cells and one third had Fc receptors, but lacked complement receptors. Although 15-30% of the cells with high K values formed rosettes with sheep erythrocytes, there was virtually no response to T-cell mitogens such as PHA and Con A. Mixing cells having high K values with mitogen-responsive T cells from intermediate cell fractions revealed no evidence of suppressor-cell activity to explain the poor mitogen response. The studies indicate that a heterogeneous population of cells share charge-related surface properties resulting in a high partition coefficient. These cells comprise a significant proportion of human peripheral blood lymphocytes and include virtually all of the large granular lymphocytes as well as a large population without this morphology that shares the feature of unresponsiveness to T-cell mitogens.     

Use of a symblepharon ring for treatment of over-filtration and leaking blebs after glaucoma filtration surgery

We report the use of a symblepharon ring in the treatment of seven cases of flat anterior chamber (six cases of total iridocorneal touch, and one of corneallens touch) secondary to overfiltration or to bleb leak. In all cases, the anterior chamber reformed within 24 hours after the symblepharon ring was used. Advantages of using the ring are: (a) it permits testing of visual acuity, tonometry, and intraocular examination without removing it; (b) it does not require suturing to the conjunctiva; (c) it does not disturb the corneal epithelium; (d) it may be available at institutions lacking other shells; and (e) it is cost-effective.     

Antibodies to beta 2-microglobulin, the light-chain component of class I histocompatibility molecules, provide an activation signal to human lymphocytes

Antibodies to beta 2-microglobulin (beta 2m), the light-chain component of class I histocompatibility antigens, provide a strong co-stimulatory signal to human lymphocytes in the presence of phorbol ester. This activation signal requires a high concentration of antibody, whereas the effect on responses to a mitogenic lectin is exclusively inhibitory over a broad dose range. A rabbit polyclonal and a mouse monoclonal antibody to beta 2m were similarly co-stimulatory. Antibody to beta 2m was co-stimulatory with phorbol ester, but not calcium ionophore, suggesting that class I antigens play a role in the initiation of the 'first' signal of a recently described two-signal model of lymphocyte activation.     

Subfractionation of human T lymphocyte subpopulations by partitioning: surface heterogeneity of the OKT8/Leu2+ T cell subset.

Human peripheral blood mononuclear cells (PBMC) were subjected to countercurrent distribution in a charge-sensitive dextran-poly (ethylene glycol) aqueous two-phase system. Cells are thereby subfractionated on the basis of their charge-associated surface properties. The helper/inducer T cell subset (OKT4/Leu3+) has a single distribution curve with a low partition ratio (P). In contrast, the suppressor/cytotoxic (OKT8/Leu2+) subset is clearly heterogeneous and gives two peaks, one with a lower and the other with a higher P value. The cells in the latter peak constitute part of a minor subpopulation of cells enriched with natural killer (Leu7+) cells. Double labelling studies indicate that a majority of Leu2+ cells with a high P value also have a marker of natural killer cells (HNK-1), whereas the Leu2+ cells with low partition ratio are depleted with respect to cells bearing both markers.     

HLA-DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland

Celiac disease (CD) has one of the strongest class II HLA associations of any human illness. We used DNA-RFLP typing to study the class II HLA genotypes of celiac disease patients from the West of Ireland, the geographic area with the highest rate of celiac disease in the world. We confirmed the high frequency of HLA-DR3 in this population, and we were also able to demonstrate the additional risk of developing celiac disease imparted by HLA-DR7. This was done by clearly distinguishing DR7, DQ2 haplotypes from DR7, DQ9 haplotypes, and by "subtraction analysis" of haplotype frequencies. As reported in other populations, most of the patients without DR3 were heterozygous for DR7 and DR11 or 12 (DR5), or had DR4. We used PCR-RFLP and direct sequencing of amplified DNA to examine HLA-DR4 subtypes. The frequency of HLA-DR4 was markedly decreased in patients compared with controls (p=0.000001) and there was a significant alteration of DR4 subtypes of the patients compared with controls (p=0.0227). Moreover, all of the CD patients (5 of 5) with DR4 had a haplotype associated with the DQB1*0302 allele compared with only 11 of 23 control subjects with DR4. Our results in this population with exceptionally high risk of CD strongly support the DQ heterodimer hypothesis and suggest that the recently described sequence difference between the DQB1*02 alleles of DR3 and DR7 may contribute to a synergistic increased risk when these haplotypes are inherited together. In addition, our findings suggest a role for HLA-DQ in DR4-associated CD.     

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F1 hybrid mice: alterations correlated with the immunodeficiency of aging

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.     

Translocation (4;11) in acute myelogenous leukemia

A child with acute myelogenous leukemia is presented. Cytogenetic analysis of her leukemic cells revealed a (4;11)(q12;q23) translocation. The slight difference in the breakpoint on chromosome #4 from previously reported cases of t(4;11) may account for the degree of myeloid differentiation expressed. Acute leukemia associated with t(4;11) is a unique subgroup that originates in an early myeloid stem cell and carries a poor prognosis.     

Partial trisomy 6p and partial monosomy 9p from a de novo translocation 46, XY, -9, + DER(9)T(6:9)(p211:p24)

This report describes an adult male with a partial trisomy 6p(p211-pter) and a partial monosomy 9p(9p24-pter) resulting from a de novo unbalanced translocation. This patient does not show the classical featured of the 9p partial monosomy syndrome, thus disputing the claim of Hoo et al. (1982) that 9p24 is the critical segment for the monosomy syndrome. Partial trisomy for 6p has only been previously reported in children. In addition to the chromosomal anomalies, the patient has autosomal recessive spinal muscular atrophy with a different age of onset than two affected sibs. Finally, he shows unusual audiologic and ophthalmologic signs nor previously reported as part of the 9p monosomy or 6p trisomy syndromes.     

The cleavage activation and sites of glycosylation in the fusion protein of Hendra virus

Hendra virus (HeV) is an unclassified member of the Paramyxoviridae family that causes systemic infections in humans, horses, cats, guinea pigs and flying foxes. The fusion protein (F(0)) of members of the Paramyxoviridae family that cause systemic infections in vivo contains a basic amino acid-rich region at which the protein is activated by cleavage into two subunits (F(1) and F(2)). HeV F(0) lacks such a domain. We have determined the cleavage site in HeV F(0) by sequencing the amino terminus of the F(1) subunit and in view of the potential effect of glycosylation on the cleavage process have ascertained the sites at which F(0) is glycosylated. The results indicate that unlike other members of the family that replicate in cultured cells and cause systemic infections in vivo, cleavage of HeV F(0) occurs at a single lysine (reside 109) in the sequence Asp-Val-Lys- downward arrow-Leu. Although HeV genotypically resembles members of the Respirovirus and Rubulavirus genera in having potential N-linked glycosylation sites in both the F(1) and F(2) subunits, we show that phenotypically HeV may more closely resemble members of the Morbillivirus genus that contain N-linked glycans only in the F(2) subunit.