Devendra P. Dubey

Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression.

Reactive oxygen species are involved in many cellular metabolic and signalling processes and are thought to have a role in disease, particularly in carcinogenesis and ageing. We have generated mice with targeted inactivation of Prdx1, a member of the peroxiredoxin family of antioxidant enzymes. Here we show that mice lacking Prdx1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe haemolytic anaemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The haemolytic anaemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, haemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of Prdx1 expression in heterozygotes, which suggests that this protein functions as a tumour suppressor. Prdx1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas Prdx1-null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate Prdx1 as an important defence against oxidants in ageing mice.

Lymphocyte subsets and integrated immune function in aging humans

Abstract Lymphocyte response to mitogen stimulation declines in aging humans. Depressed responses to phytohemagglutinin, concanavalin A, and PHA (HA-17) are evident when aged groups are compared to a young adult population. Responding capacity in the mixed lymphocyte reaction declines with age, while stimulating ability remains unaltered. The number of circulating thymus-derived (T) lymphocytes increases in the ninth and tenth decades, while lymphocyte-bearing complement receptors increase in frequency during the fifth through the eighth decades. These observations suggest that T-cell function cannot be equated solely with numbers of circulating immunocompetent cells in aging humans. We have introducted a method of multiple function analysis (pattern recognition) to classify individuals of different ages into groups according to the net-correlated immune functions. The immune functions of an aged population cluster separately from those of a young population.

A family with hereditary ataxia: HLA typing

In a previously unreported family with olivopontocerebellar atrophy, the kindred contained over 600 individuals in five generations. Of 83 offspring of affected individuals who are over 38.8 years of age (the mean age of the onset of disease in this family), 47 had ataxia; there was autosomal dominant transmission. Clinical findings included lower bulbar palsies, hyperreflexia, ataxia, incoordination, scanning and explosive speech, and, in some, slow motor-nerve conduction velocities. There was cortical and cerebellar atrophy of pontine nuclei, inferior olives, and XII nuclei, and loss of Purkinje cells in the cerebellum. Seventy-three individuals of the III and IV generations were typed for HLA histocompatibility antigens. A maximum lod score of 1.97 was found at male recombination fraction 0.18 and female recombination fraction 0.36. When the lod score values reported in other studies were combined with the values in this family, the maximum lod score was found to be 4.681 at a recombination frequency of 0.22.

Complex expression of natural killer receptor genes in single natural killer cells

Human natural killer (NK) cells express several inhibitory and non‐inhibitory NK receptors per cell. Understanding the expression patterns of these receptor genes in individual cells is important to understanding their function. Using a single‐cell reverse transcription–polymerase chain reaction (RT‐PCR) method, we analysed the expression of nine NK receptor genes in 38 resting CD56+ NK cells from peripheral blood of normal donors. We observed highly diverse patterns of receptor expression in these cells. No NK receptor is expressed universally in every CD56+ NK cell. The expressed receptor types per cell varied from two to eight. We specifically analysed the distribution of inhibitory (DL) and non‐inhibitory (DS) killer immunoglobulin‐like receptors (KIR). The frequency of individual receptor expression varied from 26% for 2DS2 to 68% for both 2DL1 and 2DL4. A comparison of the coexpression of DL and DS receptors showed a significant association in the expression of 2DL2 and 2DS2 (χ2=16·6; P<0·001) genes but no association between 2DL1 and 2DS1 or between 3DL1 and 3DS1 genes. Coexpression analysis of the 2DL1 and 2DL2 genes in 2DL4+ and 2DL4− cells showed a strong association in 2DL4+ but not in 2DL4− cells, suggesting a differential effect of the 2DL4 gene on the expression of 2DL1 and 2DL2 genes. Single‐cell RT‐PCR is a powerful tool to study multiple receptor gene expression ex vivo in individual NK cells and provides information about the expression pattern of KIR receptors that may suggest mechanisms of gene expression responsible for generation of the KIR repertoire.

Immunoglobulin deficiencies and susceptibility to infection among homozygotes and heterozygotes for C2 deficiency

About 25% of C2-deficient homozygotes have increased susceptibility to severe bacterial infections. C2-deficient homozygotes had significantly lower serum levels of IgG2, IgG4, IgD, and Factor B, significantly higher levels of IgA and IgG3 and levels of IgG1 and IgM similar to controls. Type I (28 bp deletion in C2 exon 6 on the [HLA-B18, S042, DR2] haplotype or its fragments) and type II (non-type I) C2-deficient patients with increased susceptibility to bacterial infection had significantly lower mean levels of IgG4 (p < 0.04) and IgA (p < 0.01) than those without infections (who had a higher than normal mean IgA level) but similar mean levels of other immunoglobulins and Factor B. Of 13 C2-deficient homozygotes with infections, 85% had IgG4 deficiency, compared with 64% of 25 without infections. IgD deficiency was equally extraordinarily common among infection-prone (50%) and noninfection-prone (70%) homozygous type I C2-deficient patients. IgD deficiency was also common (35%) among 31 type I C2-deficient heterozygotes (with normal or type II haplotypes), but was not found in 5 type II C2-deficient heterozygotes or 1 homozygote. Thus, C2 deficiency itself is associated with many abnormalities in serum immunoglobulin levels, some of which, such as in IgG4 and IgA, may contribute to increased susceptibility to infection. In contrast, IgD deficiency appears not to contribute to increased infections and appears to be a dominant trait determined by a gene or genes on the extended major histocompatibility complex (MHC) haplotype [HLA-B18, S042, DR2] (but probably not on type II C2-deficient haplotypes) similar to those previously identified on [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3].

Complex cytokine responses to hepatitis B surface antigen and tetanus toxoid in responders, nonresponders and subjects naive to hepatitis B surface antigen

Some human subjects vaccinated with hepatitis B surface antigen (HBsAg) do not produce antibodies to the vaccine (nonresponders). The mechanism for nonresponse is unknown. To understand the response and nonresponse to nominal antigens better, we determined the level and kinetics of cytokine secretion in response to HBsAg and tetanus toxoid (TT) by peripheral blood mononuclear cells (PBMC) in vitro from HBsAg vaccine responders and nonresponders and from individuals naive to HBsAg. Proliferating PBMC secreted peak levels of interleukin-2 (IL-2) at 2 days and peak levels of tumor necrosis factor-beta (TNF-beta), interferon-gamma (IFN-gamma), IL-4 and IL-10 at 3-6 days post-stimulation. In contrast, nonproliferating PBMC (whether from nonresponders, naive subjects or weak responders) did not produce detectable levels of TNF-beta or IFN-gamma, nor was IL-4 or IL-10 produced significantly, and that produced had a different kinetic profile from that of proliferating PBMC. HBsAg-specific cytokine production by PBMC from strong responders broadly paralleled their cytokine responses to TT. Cellular cytokine mRNA levels measured by reverse transcriptase-polymerase chain reaction corroborated the secreted cytokine results. The anti-HBsAg- and anti-TT-specific T cell cytokine responses were mixed Th(1/2)-like and donor-specific. An HBsAg-specific cytokine response, but not a TT-specific cytokine response, was completely missing in nonresponders. These data suggest that the T cell defect of HBsAg nonresponse is not due to a skewed cytokine profile.

Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity

The reproductive hormones are implicated in the well documented sexual dimorphism in cellular and immune responses. Prostaglandins (PGs) are mediators of the immune response with their concentration and relative amounts being pivotal to their impact. In resident peritoneal macrophages isolated from mice we had previously noted that the cells from females synthesized significantly more PG than males. In these experiments we investigated whether PG metabolism in the human monocyte was influenced by gender and by stage of the menstrual cycle. Monocytes isolated from the female and activated in vitro with LPS produced on average significantly more PG into the medium than the males. Among females, significantly more PG was found in the medium from cells isolated during the luteal phase of the cycle than during the early follicular phase. It was also in this luteal phase in which the female differed substantially from males. We suggest that the in vivo hormonal changes associated with the menstrual cycle modulate monocyte synthesis of PG and other immune modulators such as IL-1. This could be a key to understanding differences in vulnerability between males and females as well as within phases of the cycle, to immune and inflammatory insult.

Increased frequency of HLA-DRW4 in chronic active hepatitis.

Abstract. HLA‐A, B, C and DRw typing was performed on peripheral blood lymphocytes of 17 adults with the diagnosis of chronic active hepatitis made by liver biopsy as the only criterion for study. An increase in the frequency of HLA‐DRw4 (71 vs. 24%, p<0.005) was observed, but there was no increased frequency of HLA‐A1, B8 or any other HLA locus specificity.

The MHC influences NK and NKT cell functions associated with immune abnormalities and lifespan.

The lifespans of H-2 congenic mice differ significantly. The B10.AKM (H-2m) strain has a median survival time (MST) of 15 months, whereas the B10.BR (H-2k) strain has an MST of 24 months. It was previously shown that B10.AKM mice at 13-15 months of age have immunological function comparable to those of B10.BR mice at 22-26 months of age. These functions include: a low proliferative response, reduced levels of intracellular calcium release [Ca2+]i, and an increase in the frequency of memory helper T-cells (CD4+ CD44hiCD45RBlo). In this report similar deficiencies were demonstrated in B10.AKM mice at 2-4 months of age and show that activated spleen NK1.1+CD4+ T (NKT) cells from young B10.AKM mice produce a significantly higher level of IL-4 but a lower level of IFN-gamma as compared to NKT cells from B10.BR mice of the same age. Also, the cytotoxic activity of natural killer (NK) cells from spleens of young (2-4 months) as well as adult (12-16 months) B10.AKM mice is significantly lower (P < 0.01) than that of NK cells from B10.BR mice. These findings suggest that the NKT activity in young B10.AKM mice is a factor for the early onset of immune dysfunction leading to a shorter lifespan.

HLA-Cw7 zygosity affects the size of a subset of CD158b+ natural killer cells

Individuals with certain HLA class I genotypes are highly susceptible to disease after viral infection. Natural killer (NK) cells kill virus-infected cells through a mechanism involving HLA class I receptors. These facts may be connected if an individuals HLA genotype regulates the number and function of NK cells. We have observed that subjects homozygous for the HLA-B/C region of conserved major histocompatibility complex (MHC) extended haplotypes have lower NK cell activity and a significantly lower frequency of CD16+CD56+ NK cells than heterozygotes. The proportion of CD16−CD56+ NK cells was unaffected by zygosity for the HLA-B/C region. We show here that the frequency of CD16+CD158b+, but not CD16−CD158b+ NK cells, was significantly lower (p <0.026) in homozygotes for HLA-Cw7 (NK1 ligand) haplotypes than in heterozygotes. The frequencies of CD16+CD158a+ and CD16−CD158a+ and CD16−CD158a+ or CD16+NKB1+ and CD16−NKB1+ NK cells were not different in these donor groups. These findings suggest that the proportion of NK cells coexpressing CD16 and CD158b, but not CD158a nor NKB1, is influenced by zygosity for the HLA-Cw7 (NK1 ligand) haplotype. Since NK cells are involved in protection from virus infection, a reduced size of a ligand-specific NK subset in individuals homozygous for some HLA-B/C haplotypes may help explain their increased susceptibility to virus-induced diseases.