Functional analysis of HIV type 1 Nef gene variants from adolescent and adult survivors of perinatal infection

Throughout the world, infants and children with HIV-1 infection are increasingly surviving into adolescence and adulthood. As HIV Nef is an important determinant of the pathogenic potential of the virus, we examined nef alleles in a cohort of extreme long-term survivors of HIV infection (average age of 16.6 years) to determine if Nef defects might have contributed to patient survival. HIV nef gene sequences were amplified for phylogenetic analysis from 15 adolescents and adults infected by mother-to-child transmission (n=10) or by blood transfusion (n=5). Functional analysis was performed by inserting patient-derived nef sequences into an HIV-derived vector that permits simultaneous evaluation of the impact of the Nef protein on MHC-I and CD4 cell surface expression. We found evidence of extensive nef gene diversity, including changes in known functional domains involved in the downregulation of cell surface MHC-I and CD4. Only 3 of 15 individuals (20%) had nef alleles with a loss of the ability to downregulate either CD4 or MHC-I. Survival into adulthood with HIV infection acquired in infancy is not uniformly linked to loss of function in nef. The Nef protein remains a potential target for immunization or pharmacologic intervention.     

Male Breast Cancer: A Population-Based Comparison with Female Breast Cancer in Hong Kong,Southern China: 1997–2006

Background

Male breast cancer (MBC) is uncommon. As a result, there is limited availability of studies and reviews and even fewer reports from Asia. This is the largest population-based study to compare Chinese MBC patients with female patients during a 10-year period in Hong Kong, Southern China.

Methods

A retrospective review of medical records of 132 male and 8,118 female breast cancer patients between year 1997 and 2006 in Hong Kong was performed. Each MBC patient was matched with three female breast cancer patients for further analysis. Different characteristics, overall, breast-cancer specific, and disease-free survivals (DFS) were compared.

Results

Mean age at diagnosis of male and female patients was 64.5 and 52.7 years respectively. Male patients showed lower histological grade, overall stage, smaller tumor size, and more positive sensitivity in hormone receptors. They were more likely to die of causes other than breast cancer. Matched analysis found that the 5-year overall survival (OS), breast-cancer–specific mortality, and DFS for male and female patients were 78.7, 90.5, 90.5, and 77.9, 86.4, and 81.4 % respectively. Male patients had poorer OS at early overall stage but better breast-cancer—specific mortality rates at any age (p < 0.01). Male patients had a significant risk of dying due to any cause in the presence of distant relapse and had less risk of dying when tumor was ER-positive and HER2-positive.

Conclusions

Chinese male breast cancer patients tend to have poorer OS but better breast-cancer—specific survival compared with their female counterparts.     

Small genome of the fungus Escovopsis weberi,a specialized disease agent of ant agriculture

Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite’s genome.The highly evolved agricultural lifestyle of leaf-cutting ants has attracted particular attention because these ants cultivate a symbiotic fungus that serves as their major food source. These ants cut leaves, preprocess them into small pieces, and feed them to the cultivated fungus (1). The capacity of the cultivated fungus to break down plant material gives ant agriculturalists access to the vast nutrient stores locked within neotropical plants (Fig. 1A) (2–5). The symbiosis between fungus-growing ants and their cultivated fungi has persisted for at least 50 million years (6).Open in a separate windowFig. 1.Escovopsis weberi, a specialized mycoparasite of the fungus-growing ant symbiosis, has a small genome compared with other Pezizomycotina fungi. (A) Both fungus-growing ants and the mycoparasite E. weberi use the ants’ cultivated fungi as their primary food source. The ability of the cultivated fungi to efficiently break down plant material gives both consumers access to the biomass of neotropical plants. (B) Size and protein-coding gene content of genomes of diverse fungi in the Pezizomycotina. Bayesian phylogeny estimated using partial amino acid alignments of three genes (Rpb1, Rpb2, ef1-α). All posterior probabilities are greater than 0.95. Phylogeny is rooted with Sacchormyces cervesiae (not shown). (C) Relationship between genome size and gene content. A list of genomes included in this panel is in SI Appendix, Table S1.Like human agriculture, ant agriculture is hampered by disease. The ants’ fungal crops are attacked and consumed by fungal parasites of the genus Escovopsis (Ascomycota, Pezizomycotina: anamorphic Hypocreales) (Fig. 1A) (7), which have evolved in association with the ants and their cultivated fungi (8). Escovopsis infection can have detrimental impacts on garden health and, consequently, on the survival of ant colonies (9, 10). Such mycoparasitism, the phenomenon whereby one fungus is parasitic on another fungus, is rare. It is most well-known for species from the genus Trichoderma, some of which are used as biocontrol agents for fungal diseases and others of which attack human-cultivated fungi (11–13). In contrast to Trichoderma species, however, Escovopsis species grow poorly in their hosts’ absence (SI Appendix, Figs. S1 and S2).Escovopsis species have never been isolated outside of fungus-growing ant colonies, and different strains of Escovopsis are capable of attacking the fungi grown by different fungus-growing ant species (8, 14, 15). The long-term, specialized evolutionary history of the association between Escovopsis and their hosts provides a unique venue to explore the consequences of host specialization on pathogen genome evolution. Here, we assemble and annotate the genome of a strain of Escovopsis weberi. Consistent with expectations under an evolutionary transition toward using a narrow host range, and similar to many other specialized, host-associated microbes (16, 17), E. weberi exhibits gene loss. Contrasting other fungal pathogens, the large genomes of which are expanded with genetic elements that influence host adaptation (18), the genome size of Escovopsis is small compared with those of its closest sequenced relatives.     

Interleukin-4 and interleukin-5 map to human chromosome 5 in a region encoding growth factors and receptors and are deleted in myeloid leukemias with a del(5q)

Interleukin-4 (IL-4) is a potent mediator of growth and differentiation of cells of several hematopoietic lineages. Interleukin-5 (IL-5) is a lineage-specific hematopoietic growth factor that stimulates the production of eosinophils and eosinophil colonies from normal human bone marrow cells. By using somatic cell hybrids and in situ chromosomal hybridization, we localized the IL-4 and IL-5 genes to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, the IL-4 and IL-5 genes were found to be deleted in the 5q- chromosome of four patients with refractory anemia (RA) or therapy-related acute nonlymphocytic leukemia (t-ANLL), who had a del(5q). Thus a small segment of chromosome 5 contains IL-4, IL-5, IL- 3, and GM-CSF as well as other genes such as CD14 and EGR1. Our findings that each of these genes was deleted in the 5q- chromosome suggest that loss of function of one or more of these genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).     

Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor

To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.     

Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic diseases

Translocations and deletions of the short arm of chromosome 12 [t(12p) and del(12p)] are common recurring abnormalities in a broad spectrum of hematologic malignant diseases. We studied 20 patients and one cell line whose cells contained 12p13 translocations and/or 12p deletions using fluorescence in situ hybridization (FISH) with phage, plasmid, and cosmid probes that we previously mapped and ordered on 12p12-13. FISH analysis showed that the 12p13 translocation breakpoints were clustered between two cosmids, D12S133 and D12S142, in 11 of 12 patients and in one cell line. FISH analysis of 11 patients with deletions demonstrated that the deletions were interstitial rather than terminal and that the distal part of 12p12, including the GDI-D4 gene and D12S54 marker, was deleted in all 11 patients. Moreover, FISH analysis showed that cells from 3 of these patients contained both a del(12p) and a 12p13 translocation and that the affected regions of these rearrangements appeared to overlap. We identified three yeast artificial chromosome (YAC) clones that span all the 12p13 translocation breakpoints mapped between D12S133 and D12S142. They have inserts of human DNA between 1.39 and 1.67 Mb. Because the region between D12S133 and D12S142 also represents the telomeric border of the smallest commonly deleted region of 12p, we also studied patients with a del(12p) using these YACs. The smallest YAC, 964c10, was deleted in 8 of 9 patients studied. In the other patient, the YAC labeled the del(12p) chromosome more weakly than the normal chromosome 12, suggesting that a part of the YAC was deleted. Thus, most 12p13 translocation breakpoints were clustered within the sequences contained in the 1.39 Mb YAC and this YAC appears to include the telomeric border of the smallest commonly deleted region. Whether the same gene is involved in both the translocations and deletions is presently unknown.     

Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.