Absence Of Lymphocytes Research Articles

Quantitative Analysis of the P65 Gene Expression in Patients with Coloroctal Cancer

P65 gene expression level was determined in colon cancer cases by means of real-time PCR. 51 cases of colorectal carcinomas showing positive RT-PCR signals for P65 gene expression selected from 109 frozen samples were further investigated by quantitative real-time PCR. P65 levels were higher in cancer with metastases to lymph nodes and distant metastases. Higher levels were observed in more advanced cases classified as III and IV according to pTNM classification. In two groups of patients with vessel invasion and absence of lymphocytes in tumour tissue, the presence of P65 expression correlated with shorter survival time. Quantitative results confirmed that P65 gene expression in colon cancer is engaged in the process of metastasis formation and could be correlated with worse prognosis for the patients.

Essential crosstalk between myeloid and lymphoid cells for development of chronic colitis in myeloid‐specific signal transducer and activator of transcription 3‐deficient mice

Dysregulated cytokine responsiveness by myeloid cells can be a trigger for the development of chronic inflammation as well as inflammatory bowel disease. Thus, mice with a myeloid-specific defect in signal transducer and activator of transcription (Stat) 3 develop spontaneous colitis secondary to the inability of myeloid cells to respond to the immunosuppressive cytokine interleukin-10. We now examined whether the inflammation caused by Stat3-deficient macrophages is cell autonomous or dependent on their interaction with lymphocytes. For this purpose, myeloid-specific Stat3-deficient mice (LysMcre/Stat3(flox) mice) were intercrossed with RAG-1 knockout mice to generate LysMcre/Stat3(flox) RAG(-/-) mice. In these mutants and LysMcre/Stat3(flox) littermate control mice we determined the onset and severity of spontaneous chronic enterocolitis, and the reaction to dextran sodium sulphate (DSS)-induced epithelial damage, as well as to lipopolysaccharide (LPS) challenge. In contrast to LysMcre/Stat3(flox) mice, LysMcre/Stat3(flox) RAG(-/-) animals are protected from chronic enterocolitis. Although they respond to oral dextran sulphate with acute colitis symptoms, the inflammation heals similarly to wild type mice whereas LysMcre/Stat3(flox) mice exhibit continued colitis pathology. In addition, the hyperreactivity of LysMcre/Stat3(flox) mice to LPS-challenge in vivo was less severe in the absence of lymphocytes. Despite clear differences in the strength of inflammatory responses, macrophages of both LysMcre/Stat3(flox) mice and LysMcre/Stat3(flox) RAG(-/-) animals exhibited increased costimulatory capacity. In conclusion, our findings demonstrate that Stat3-deficient myeloid cells alone are not capable of inducing the severe pathology seen in LysMcre/Stat3(flox) mice. Yet when these cells can interact with lymphocytes their increased costimulatory potential will trigger an overshooting inflammatory response.

Preoperative Diagnosis of Extrapancreatic Neural Invasion in Pancreatic Cancer

Pancreatic cancer recurs in most patients after resection with curative intent. Recurrence is particularly common in patients with extrapancreatic neural invasion (EPNI), the presence of which correlates with poor prognosis. Macroscopic EPNI may be detected with conventional noninvasive imaging and endoscopic ultrasound (EUS) imaging, but microscopic EPNI has required postoperative pathologic examination of surgical specimens. We report the preoperative diagnosis of cancer infiltration into celiac ganglia. We hypothesized that microscopic pancreatic cancer metastasis to neural ganglia can be detected by EUS-guided biopsy examination. We performed a retrospective review of patients with pancreatic cancer undergoing EUS in whom celiac ganglia were sampled to exclude malignant infiltration. Six patients with pancreatic cancer underwent EUS-guided fine-needle aspiration or trucut biopsy examination of presumed celiac ganglia. Metastatic cancer was found in ganglia of 2 patients. Specimen review identified adenocarcinoma and neural tissue in the absence of lymphocytes. At laparoscopy, 1 of the 2 patients with positive celiac biopsy specimens also had several unexpected peritoneal metastatic deposits. The other patient was considered to have locally advanced unresectable disease. Both patients are receiving supportive care. EPNI may be shown preoperatively in patients with pancreatic cancer using EUS-guided sampling of celiac ganglia. A preoperative diagnosis of EPNI has the potential to improve staging accuracy and patient outcomes.

Fine-needle aspiration cytology of pancreatic lymphoepithelial cysts

Lymphoepithelial cysts (LECs) of the pancreas are extremely rare, benign, nonneoplastic cysts that can mimic pseudocysts or cystic neoplasms clinically and radiographically. The cytologic features of LECs have been described only in a handful of case reports and may overlap with both benign and malignant pancreatic tumors. The authors conducted a 5-year, retrospective, computerized review of the cytopathology files of 2 institutions for all diagnoses of pancreatic LECs. Clinical, radiographic, cytologic, and chemical findings were reviewed. Four patients were identified. The study group consisted of 3 men and 1 woman who ranged in age from 33 years to 63 years. The masses were located throughout the pancreas. Tumor sizes ranged from 1.8 cm to 5.7 cm in greatest dimension. Smears from all patients revealed numerous anucleated squamous cells, rare benign nucleated cells, amorphous debris, and an absence of lymphocytes. Mildly atypical mucinous glandular and parakeratotic epithelium were identified in 2 patients, leading to diagnoses of atypical and suspicious for malignancy. Subsequent surgical follow-up of 3 patients revealed pancreatic LECs. Pancreatic LECs are extremely rare, and certain cytologic pitfalls may hinder a correct prospective diagnosis. Familiarity with the lesion's clinical and cytologic features may help clinicians arrive at the appropriate prospective diagnosis and, thus, permit conservative management. Cancer

Mutation of mouse Mayp/Pstpip2 causes a macrophage autoinflammatory disease

AbstractMacrophage actin-associated tyrosine phosphorylated protein (MAYP)/PSTPIP2, a PCH protein, is involved in the regulation of macrophage motility. Mutations in a closely related gene, PSTPIP1/CD2BP1, cause a dominantly inherited autoinflammatory disorder known as PAPA syndrome. A mutant mouse obtained by chemical mutagenesis exhibited an autoinflammatory disorder characterized by macrophage infiltration and inflammation, leading to osteolysis and necrosis in paws and necrosis of ears. Positional cloning of this recessive mutation, termed Lupo, identified a T to A nucleotide exchange leading to an amino acid substitution (I282N) in the sequence of MAYP. MaypLp/Lp disease was transferable by bone marrow transplantation and developed in the absence of lymphocytes. Consistent with the involvement of macrophages, lesion development could be prevented by the administration of clodronate liposomes. MAYP is expressed in monocytes/macrophages and in a Mac1+ subfraction of granulocytes. LPS stimulation increases its expression in macrophages. Because of the instability of the mutant protein, MAYP expression is reduced 3-fold in MaypLp/Lp macrophages and, on LPS stimulation, does not rise above the level of unstimulated wild-type (WT) cells. MaypLp/Lp mice expressed elevated circulating levels of several cytokines, including MCP-1; their macrophages exhibited altered cytokine production in vitro. These studies suggest that MAYP plays an anti-inflammatory role in macrophages.

Mutation of Mouse MAYP/PSTPIP2 Causes a Macrophage Autoinflammatory Disease.

Macrophage actin-associated tyrosine phosphorylated protein (MAYP), a Pombe Cdc15 Homology (PCH) family protein also known as proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), is involved in the regulation of macrophage motility (Chitu et al., 2005). Mutations in a closely related gene, PSTPIP1/CD2 binding protein 1 (CD2BP1) cause a dominantly inherited autoinflammatory disorder known as PAPA syndrome. A mutant mouse obtained by chemical mutagenesis exhibited an autoinflammatory disorder characterized by macrophage infiltration and inflammation leading to osteolysis and necrosis in paws and necrosis of ears. Positional cloning of this recessive mutation, termed Lupo, identified a T to A nucleotide exchange leading to an amino acid substitution (I282N) in the sequence of MAYP. MaypLp/Lp disease was transferable by bone marrow transplantation and developed in the absence of lymphocytes. Consistent with the involvement of macrophages, lesion development could be prevented by the administration of chlodronate liposomes. MAYP is expressed in monocytes/macrophages and a Mac1+ sub-fraction of granulocytes. LPS stimulation increases its expression in macrophages. Due to instability of the mutant protein, MAYP expression is reduced 3-fold in MaypLp/Lp macrophages and upon LPS stimulation does not rise above the level of unstimulated wt cells. MaypLp/Lp mice expressed elevated circulating levels of IL-4, Rantes, TGF-beta and MCP-1 and in vitro their macrophages exhibit altered cytokine production. These studies suggest that MAYP plays an anti-inflammatory role in macrophages.

Identification of Two Molecular Groups of Seminomas by Using Expression and Tissue Microarrays

Highly effective tailored clinical management of testicular germ cell tumors is based on the identification of two major histologic subtypes: seminomatous and nonseminomatous germ cell tumors. Expression array analysis of these two histologic subtypes using hierarchical clustering reveals two tumor groups, one composed solely of seminomas and the other containing embryonal carcinomas and seminomas. Supervised analysis between these groups identified 55 significantly dysregulated genes (false discovery rate = 2.3). The genes with the highest overexpression in the first group compared with the second included SLC43A1 (POV1), NET-7, IGF2, and JUP; down-regulated genes included GRB7, PFKP, and CDC6. In situ hybridization of SLC43A1 mRNA showed significantly increased signal intensity in the seminomas. At the protein level, expression of the immunohistochemical markers cytokeratins (pan-cytokeratin staining), placental-like alkaline phosphatase, anti-cytokeratin clone 5.2, CD30, anion exchanger 1/3, junction plakoglobulin (JUP), and POU domain, class 5, transcription factor 1 (octomer-binding transcription factor 3/4) was significantly different between seminomas and embryonal tumors. Hierarchical clustering based on a refined protein expression profile identified two groups, the first consisting solely of seminomas the other of seminomas and embryonal carcinomas. No histomorphologic differences were observed between the two seminoma groups such as the presence or absence of lymphocytes or extent of stromal elements. In summary, using independent methodologies and samples, we have identified two groups of seminomas. One group of seminomas has a molecular profile similar to embryonal carcinoma. The findings in the current study may help explain aberrant immunoprofiles seen with some seminomas.

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Our study1 referred to by Professor Pender describes a very early stage in the formation of new multiple sclerosis (MS) lesions characterized by the presence of broad areas of palely staining (but still largely intact) myelin sheaths, containing apoptotic oligodendrocytes and activated microglia, but few or no myelin phagocytes or lymphocytes. The absence of T lymphocytes in the immediate vicinity of apoptotic oligodendrocytes is especially significant, because such changes have not been reported in any form of the animal model, experimental allergic encephalomyelitis (EAE). As noted by ourselves and Dr Trapp,2 this raises the possibility of important differences in the pathogenesis of these two diseases. Professor Pender suggests that steroid administration shortly before death may have cleared the central nervous system (CNS) of T cells; this possibility can be ruled out first by the fact that not all seven patients (in whom apoptotic lesions were identified) received such treatment, and second by the presence of numerous lymphocytes in zones of active phagocytosis adjacent to or contiguous with areas exhibiting prominent oligodendrocyte apoptosis in each of the 10 apoptotic lesions described. In the patient with the 17-hour symptomatic lesion, we described an absence of lymphocytes in zones of oligodendrocyte apoptosis, but relatively numerous CD4, CD8, and especially CD45RO+ T cells together with myelin phagocytes elsewhere in the lesion.1 Also as noted in our study, the findings were consistent in each of the nine other apoptotic lesions described. Our findings argue strongly against direct T-cell–mediated destruction of oligodendrocytes in early MS lesions. Rather, they suggest that dissolution of myelin by macrophages is largely or entirely scavenging activity directed against dead myelin sheaths, that is, plasma membrane of apoptotic oligodendrocytes. As noted by Professor Pender, however, our results do not rule out antibody or other humoral factors as effectors of oligodendrocyte apoptosis in early MS lesions. John W. Prineas MB, BS*, Michael H. Barnett MB, BS*, * Department of Medicine, The University of Sydney, Sydney, Australia.

Roles of lymphoid cells in the differentiation of Langerhans dendritic cells in mice

Langerhans dendritic cells are antigen presenting cells (APC) that reside within the epidermis and are capable of stimulating naive T cells. Reciprocally, lymphocytes may play a role in Langerhans cells (LC) differentiation. Our results show that the differentiation of skin LC is unaffected in the absence of lymphocytes and/or signaling through the common cytokine receptor γ chain ( γ c) required for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 signaling. Migration of LC and other dendritic cells (DC) from the skin to the draining lymph nodes (LNs) after FITC skin sensitization, is unaffected in the absence of lymphocytes or CD40. FITC + LC/DC sorted from the LNs of lymphoid deficient or control mice stimulated naı̈ve T cells with similar efficiency. However, while the absence of lymphocytes did not appear to affect the phenotype or number of emigrating LN DC/LC, their persistence in the LN appears to depend on αβ T cells. Thus, DC are strikingly reduced in numbers in the peripheral LNs of T-cell deficient mice. Finally, CD8 α expression on skin emigrants was low and dependent on the presence of CD8 + lymphocytes, while spleen CD8 + DC were present in the absence of lymphocytes. We conclude that the presence of T cells is not required for the differentiation and migration of resident skin DC but is critical for the maintenance of DC and LC migrating into the LNs.

Establishment of early lymphoid organ infrastructure in transplanted tumors mediated by local production of lymphotoxin alpha and in the combined absence of functional B and T cells.

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin alpha (LTalpha) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTalpha is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTalpha-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTalpha in the absence of lymphocytes. A CD4(+)CD3(-)CD11c(+) cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTalpha. Furthermore, in nude mice, B cells migrated in response to LTalpha and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTalpha in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTalpha is a critically important cytokine for formation of lymphoid organ infrastructure.

Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization.

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t(1/2). Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

Compartmentalization of Peyer's patch anlagen before lymphocyte entry.

We have shown that Peyer's patch (PP) first develops as a simple and even cell aggregation during embryogenesis. To investigate when and how such a simple cell aggregation forms the complex PP architecture, we analyzed the distribution of cells expressing IL-7R alpha (PP inducer cells), VCAM-1 (mesenchymal cells), CD11c (dendritic cells), and mature lymphocytes by whole-mount immunostaining of 17.5 days post coitus to 2 days postpartum mouse gut. Our results show that compartmentalization of PP anlagen commences at day 18.5 of gestation by clustering and subsequent follicle formation of IL-7R alpha(+), VCAM-1(+), and CD11c(+) cells. This process adds the primitive architecture of PP anlage with several follicles in which IL-7R alpha(+) cells localize in the center, while VCAM-1(+) and CD11c(+) cells localize at the fringe. This follicle formation is accompanied by the establishment of PP-specific vascular network expressing mucosal addressin cellular adhesion molecule-1. Mature B and T lymphocytes entering in the PP anlage are distributed promptly to their own target zones; B cells to the follicle and T cells to nonfollicular zones. Our analysis of scid/scid mouse indicate that the initial processes including formation of PP-specific vascular network occur in the absence of lymphocytes. These observations indicate that the basic architecture of PP is formed by a set of cell lineages assembled during the initial phase of induction of PP anlagen before entry of mature lymphocytes.

Cytogenetic and Immunohistochemical Characterization of Fragile X Syndrome in a Kuwaiti Family: Rapid Antibody Test for the Diagnosis of Mental Retardation Patients

Objectives: To study a Kuwaiti family with fragile X mental retardation syndrome and present a rapid, noninvasive antibody test to be used for fragile X syndrome diagnosis on all mentally retarded patients in Kuwait. Methods: For cytogenetic analysis, the fragile site was induced by culturing blood lymphocytes in a medium containing a low concentration of folate (M199) or by adding 0.1 µM of fluorodeoxyuridine to the culture media. For the immunohistochemical test on blood smears, monoclonal antibodies against fragile X mental retardation (FMR1) gene product were used. The test is based on the presence of the protein in lymphocytes from normal individuals and its absence in lymphocytes from fragile X patients. Results: A Kuwaiti family with fragile X syndrome in 4 affected males is described. The immunohistochemical study with the monoclonal antibodies against FMR protein gave conclusive results on the same day for the affected males. Conclusion: To our knowledge, this is the first report describing a large Kuwaiti family with the fragile X syndrome. These data suggest that the immunohistochemical test used is an ideal method for the diagnosis of fragile X syndrome in mentally retarded patients in Kuwait.

Two Different Types of Sialoadenitis in the NOD- and MRL/lpr Mouse Models for Sjögren's Syndrome: A Differential Role for Dendritic Cells in the Initiation of Sialoadenitis?

Sjögren's syndrome is an autoimmune disease that primarily affects the salivary and lacrimal glands. In these glands, focal lymphocytic infiltrates develop. Little is known about the initiation of this autoimmune disease. Antigen-presenting cells (APC) such as dendritic cells (DC) can play a role in the initiation of autoimmunity. To date, no data on the presence of DC in Sjögren's syndrome are available. Several mouse strains, the nonobese diabetic (NOD) and the MRL/lpr mouse, can be used as models for Sjögren's syndrome. We compared the development of sialoadenitis in the submandibular glands (SMG) of NOD and MRL/lpr mice with particular focus on the presence of APC. DC, macrophages, T cells, and B cells in the SMG were studied by means of immunohistochemistry, after which positively stained cells were quantified. NOD-severe combined immunodeficiency (SCID) mice were used to study the presence of APC in the SMG in the absence of lymphocytes. Before lymphocytic infiltration, increased numbers of DC were detected in the SMG of NOD mice compared with those numbers in control mice and MRL/lpr mice, which suggests that DC play a role in the initiation of sialoadenitis in NOD mice. In the SMG of NOD mice, lymphocytic infiltrates organized in time. In MRL/lpr mice, however, lymphocytic infiltrates were already organized at the time of appearance. This organization was lost over time. In conclusion, two types of sialoadenitis are described in two mouse models for Sjögren's syndrome. Differences exist with regard to early events that may lead to the development of sialoadenitis and to the composition and organization of inflammatory infiltrates. It is possible that different types of sialoadenitis also exist in humans and that the pathogenetic process in both the early and late phases of the autoimmune reaction differs among patients.

Down regulation of CD4 expression following isolation and culture of human monocytes.

The down regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric experiments were done to examine which factors might influence this phenomenon. The addition of lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression routinely observed following overnight culture. The down regulation was an adherence-independent phenomenon and was not influenced by the type of anticoagulant into which the peripheral blood was collected or by the presence or absence of lymphocytes within the cultures. The avoidance of the use of Ficoll-Paque to isolate peripheral blood mononuclear cells did not prevent monocyte CD4 down regulation. Finally, by tagging monocyte CD4 with an anti-CD4 phycoerythrin-conjugated monoclonal antibody prior to culture, we were able to determine that the down regulation observed was the result of the internalization of the molecule. At this time, we conclude that the observed down regulation of monocyte CD4 is probably due to the differentiation of blood monocytes into tissue culture-derived macrophages rather than to some artifact of the isolation procedure.

Fibroblast biology: Signals targeting the synovial fibroblast in arthritis

Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.

Hydrophilicity of polymers and soft tissue responses: a quantitative analysis.

This study investigates the effect of changing the hydrophilicity of polyurethane on the soft tissue inflammatory response after implantation into rats. A series of five polyurethanes were made from the macrodiols poly(ethylene oxide) (PEO), poly(tetramethylene oxide) (PTMO), poly (hexamethylene oxide) (PHMO), poly(octamethylene oxide) (POMO), and poly(decamethylene oxide) (PDMO). In the sequence the macrodiols become less polar and phase separation in the polymer increases, creating a range of hydrophilicity. These five polyurethanes were studied along with commercial Pellethane 2363-80A. The materials were implanted intramuscularly into rats for 7 days, 1 month, and 3 months. The inflammatory response was analyzed using a combination of immunohistochemistry, enzyme histochemistry, and image analysis to determine the specific cell types involved in the response and to quantify these cellular responses. The presence of three cytokines also was investigated. The cell types targeted were neutrophils, monocytes, macrophages, and T and B lymphocytes. The cytokines target were tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). All samples were positive for macrophages and activated macrophages, TNF alpha, and MCP-1, and negative for all other antibodies. The response was sustained throughout the implantation period with no significant difference among the samples except at the 7-day time point. The study demonstrated an absence of lymphocytes and neutrophils in a response that was sustained in terms of macrophages with the presence of TNF alpha and MCP-1 and the absence of IL-6. The position of subsets of macrophages with respect to increasing distance from the implants was demonstrated to be significant and consistent within this series of polyurethanes.

Macrophage tropism: fact or fiction?

Primary HIV-1 isolates can be distinguished by phenotypic qualities such as the ability to productively infect cells of established CD4-positive lines and to induce syncytia in MT-2 cells. Such viral phenotypes have also been reported to confer host cell specificity. It is perceived that primary isolates with the syncytium-inducing phenotype (SI or rapid/high) are T cell tropic and are therefore unable to infect primary cells of the monocyte/macrophage lineage. However, we have consistently found that these isolates are as capable of establishing infection in monocyte-derived macrophages (MDM) as the monocytotropic, non-syncytium-inducing variants (NSI or slow/low). It is known that differentiation, activation, and proliferation of human monocytes are affected by both isolation methods and culture conditions. Therefore, to test whether our inability to discriminate macrophage tropic HIV-1 isolates could be explained by differences in culturing techniques, we isolated monocytes by elutriation or short-term adherence and allowed the cells to mature and differentiate in either the presence or absence of autologous lymphocytes. After removal of nonadherent cells, MDM were infected with a panel of SI and NSI primary HIV-1 isolates. MDM were susceptible to infection by the SI as well as the NSI isolates, regardless of whether or not the cells were allowed to mature in the presence of autologous lymphocytes. However, MDM matured in the presence of autologous lymphocytes replicated HIV-1 isolates (both NSI and SI) to a higher titre than MDM matured in the absence of lymphocytes. In light of these findings and recently published reports on HIV-1 phenotype and chemokine receptor usage we believe that the term macrophage-tropic strains of HIV-1 is no longer appropriate.

Lymphocytes from CML patients lack a 47 kDa factor having affinity for a genomic sterol regulatory sequence

Deranged cellular cholesterol homeostasis has been widely recognized in the initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Since the human genomic sterol regulatory element (SRE) has been shown to regulate various key genes involved in this phenomenon, the present study revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects, as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when these CML patients achieved complete haematological remission (CHR) through α-interferon therapy. Furthermore, an inverse relationship was also observed between the LDL receptor gene expression at the transcriptional level and the binding affinity of this 47 kDa protein factor to the SRE sequence. Based upon these results we propose that this factor may have a role in pathophysiology of chronic myeloid leukaemia.

Sterol dependent LDL-receptor gene transcription in lymphocytes from normal and CML patients.

Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients.