Immunocytochemistry and in situ hybridisation of epidermal growth factor receptor and relation to prognostic factors in breast cancer.

The breast tumour distribution of epidermal growth factor receptor (EGFR) was studied in 193 patients with primary breast cancer by immunocytochemistry on frozen sections. EGFR was correlated (P = 0.0009) with growth fraction assessed by Ki-67, and negatively correlated with oestrogen receptor (ER, P = 0.0001) and progesterone receptor (PR, P = 0.0001) status. In 47 patients, in-situ hybridisation for EGFR mRNA showed good agreement with the immunocytochemically assessed EGFR protein. There were, however, several tumours in which EGFR mRNA could be detected in the absence of EGFR protein and there were differences between the ER and PR status of those tumours in which translation of EGFR mRNA was not seen. The cause of these differences is unclear, but these findings may represent a clue as to the differential control of breast cancer cell receptors.

Variations in CD4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus.

The expression of CD4 antigen on the surface of LeuM3-positive human blood monocytes was found to be variable with 65 to 90% of cells from 46 normal human volunteers being positive by dual staining flow cytometry. When monocytes adhered to plastic (but not when cultured on Teflon), a marked decrease in CD4 expression was observed between 1 and 24 h post-adherence. CD4 expression could not be detected in macrophages adhered to plastic for 5 days by using four anti-CD4 monoclonal antibodies in flow cytometry or direct immunofluorescence. Conversely an increasing proportion of adherent cells expressed LeuM3 and OKM5 surface antigens over the 5 days. CD4 mRNA levels were measured by slot-blot and Northern hybridization, and total cellular CD4 protein levels by immunoprecipitation. Both cellular mRNA and CD4 levels remained constant throughout the 5 day period but membrane CD4 protein levels were greatly reduced indicating that the down-regulation of CD4 was post-translational. Infection with two of six fresh human immunodeficiency virus (HIV) isolates showed different kinetic patterns when tested on purified monocytes recently adhered to plastic and macrophages adherent for 5 days. HIV antigen and reverse transcriptase levels in infected monocyte cultures remained high for 3 to 4 weeks before detachment and necrosis of the cells occurred. Infection of macrophages generated much lower levels of antigen and reverse transcriptase which declined to very low or undetectable levels over 2 weeks, leaving persisting viable macrophages. One week after infection HIV nucleic acid was detected in 69 +/- 7% of monocytes and 6 +/- 3% of macrophages by in situ hybridization. Blocking experiments with anti-Leu3a monoclonal antibody suggested that HIV infection of 5 day adherent macrophages occurred mainly by a mechanism other than binding to CD4.

Chronic colitis associated with human immunodeficiency virus infection.

OBJECTIVE: A clinical and pathological description of chronic colitis associated with human immunodeficiency virus (HIV) infection. DESIGN: A retrospective case review. SETTING: Tertiary referral institution and specialist gastroenterology practice. PATIENTS: A series of six patients with human immunodeficiency virus infection and chronic colitis observed for up to four years. RESULTS: The six patients had chronic diarrhoea for longer than six months, rectal bleeding, abdominal pain and stool leukocytosis. The mucosal pattern on colonoscopy showed diffuse proctocolitis, consisting of contact bleeding, superficial ulcerations, exudates, and/or loss of vascular pattern. Colonic biopsies showed a persisting diffuse colitis characterised principally by a mixed inflammatory cell infiltrate (mononuclear cells and neutrophils) and essentially preserved crypt architecture after six to 39 months of histological follow-up. The histopathology over this time frame was not typical of ulcerative colitis or Crohn's disease, nor of the conventionally described forms of infective colitis. HIV nucleic acid was identified by insitu hybridisation in colonic biopsies from four patients. In two of three patients tested, the presence of HIV DNA was confirmed by Southern blot analysis. No other microbial agent could be demonstrated as the cause of diarrhoea. At presentation all patients had CD4+ lymphocyte counts greater than 265 x 10(6)/L and none had the acquired immunodeficiency syndrome. Four patients have gone into remission, the other two patients were not in remission at four and a half to five years after onset. CONCLUSION: We suggest that chronic colitis may represent a new entity related to infection of the colon with human immunodeficiency virus.

The state of maturation of monocytes into macrophages determines the effects of IL-4 and IL-13 on HIV replication.

The molecular mechanisms of the effects of IL-4 and IL-13 on HIV infection in human monocytes as they matured into monocyte-derived macrophages over 7 days were investigated using HIV-1(BaL), and low passage clinical strains. IL-4 and IL-13 up-regulated the expression of both genomic and spliced HIV mRNA in monocytes cultured on Teflon, as determined by Northern analysis and p24 Ag assay. Using a nuclear run-on assay, IL-4 stimulation was shown to enhance transcription by two- to threefold. IL-4 stimulated nuclear factor-kappaB nuclear translocation and binding before enhancement of HIV RNA expression. Conversely, IL-4 and IL-13 markedly and significantly inhibited HIV replication at the transcriptional level in monocyte-derived macrophages, and this occurred whether these cytokines were added before or after HIV infection. The reversal from stimulation to inhibition occurred after 3 to 5 days of adherence to plastic. IL-4 had no significant effect on HIV reverse transcription. The effect of both cytokines on the monocyte maturation/differentiation (CD11b, CD13, and CD26) and other macrophage markers (CD14 and CD68) was examined. IL-4 enhanced CD11b, but inhibited CD26 expression and delayed CD13 loss. IL-13 had similar effects on CD11b and CD13, but no effect on CD26. Hence, these cytokines do not simply enhance monocyte differentiation, but have complex and slightly divergent effects that impact on HIV replication probably through cell signaling pathways and nuclear factor-kappaB translocation.

Recombinant interleukin 4 stimulates human immunodeficiency virus production by infected monocytes and macrophages.

Recombinant interleukin 4 (IL-4) stimulated extracellular (EC) and intracellular (IC) production of human immunodeficiency virus (HIV) from infected human blood-derived monocytes and macrophages when incubated with the cells after but not before virus inoculation. Significant stimulation was observed in 20 of 27 experiments with monocytes (inoculated with HIV immediately after adherence) and 10 of 13 experiments with macrophages (inoculated after 5 days adherence) using a total of 30 normal donors of monocytes and macrophages, and 11 recent isolates of monocytotropic HIV strains (after one passage in mononuclear cells). Marked increases in EC and IC HIV antigen were observed in some experiments, which were comparable with the maximal stimulatory effects of other cytokines such as IL-2. IL-4 also had similar effects on infectious HIV concentration as measured by reverse transcriptase and TCID50 assays. Antibody to IL-4 prevented the stimulatory effect of the cytokine. The proportion of monocytes and macrophages infected by HIV, as determined by in situ hybridization, also increased after incubation with IL-4 for 7 days. The most marked effects were observed with HIV-infected macrophages, for which the proportion of unstimulated infected cells was lower (35 to 45% increasing to 66 to 70% with IL-4 treatment). There was also an increased proportion of cells with high granule concentrations, suggesting that IL-4 increases the intracellular concentration of viral nucleic acids. This was supported by semi-quantitative hybridization experiments showing that total HIV RNA increased in IL-4-stimulated monocytes 48 to 96 h after HIV inoculation. A marked increase in aggregates was observed on day 7 in HIV-infected monocytes treated with IL-4, compared to that in HIV-infected cells alone or IL-4-treated uninfected monocytes. These findings suggest that IL-4 stimulates HIV replication in the early phases of infection and may also facilitate virus transmission by aggregate formation.

Human immunodeficiency virus type 1 infection of human placental macrophages in vitro.

Placental macrophages (Hofbauer cells) were isolated and cultured in vitro to investigate their susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Of adherent cells, 80% expressed CD14, and > 99% were nonspecific esterase-positive. CD4 antigen was expressed at very low levels. CD4 mRNA could be detected in the cells by reverse transcription followed by polymerase chain reaction. The macrophages were infected productively after inoculation with low-passage blood isolates of cell-free HIV-1. Peak virus titers were detected 3-7 days after infection by HIV-1 antigen ELISA and reverse transcriptase assay. Replication of HIV-1 in placental macrophages was less than in blood monocytes. HIV-1 RNA was detected in placental macrophages by in situ hybridization 16 days after infection. Multinucleated giant cells were identified in some cultures, indicative of an HIV-induced cytopathic effect. Thus, placental macrophages can be infected productively with clinical isolates of HIV-1, and such cells may act as a reservoir of virus for transmission to the fetus in utero.