Expression of EpCAM is up-regulated during regeneration of renal epithelia.

The human epithelial cell adhesion molecule (hEpCAM) is involved in epithelial morphogenesis and repair of epithelial tissues. We hypothesized that changes in hEpCAM expression in vivo correlate with regeneration of renal epithelia after ischaemia/reperfusion injury (IRi). Unilateral IRi was performed on kidneys of hEpCAM transgenic mice. Changes in hEpCAM expression were investigated by quantitative RT-PCR in renal cortex and medulla dissected by laser dissection microscopy and expression patterns of hEpCAM in regenerating kidneys were assessed by immunohistochemistry. The mechanism of hEpCAM promoter activation was investigated in vitro, by real-time bioluminescent imaging in HK-2 cells and in primary tubular epithelial cells (PTECs) subjected to hypoxia and reoxygenation. In vivo, the transcription of the human epcam gene significantly increased in the renal cortex during tubular re-epithelialization (p < 0.01). Moreover, the number of tubuli that expressed hEpCAM protein more than doubled in the renal cortex during regeneration. De novo expression of hEpCAM was detected in the S1 segments of proximal tubuli. Under hypoxic conditions in vitro, activity of the hEpCAM promoter was up-regulated two-fold in the HK-2 proximal epithelial cell line. Moreover, both in primary proximal epithelial cells and in HK-2 cells, hEpCAM protein expression was increased after hypoxia and reoxygenation. The significant up-regulation of hEpCAM during post-ischaemic renal regeneration in vivo and during in vitro hypoxia indicates that hEpCAM expression is associated with renal regeneration.

Pompe disease: current state of treatment modalities and animal models.

Pompe disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of acid-alpha-glucosidase (GAA). This deficiency results in glycogen accumulation in the lysosomes, leading to lysosomal swelling, cellular damage and organ dysfunction. In early-onset patients (the classical infantile form and juvenile form) this glycogen accumulation leads to death. The only therapy clinically available is enzyme replacement therapy, which compensates for the missing enzyme by i.v. administration of recombinant produced enzyme. The development of clinically relevant animal models gained more insight in the disease and allowed evaluation of recombinant enzyme therapy. Several therapies are currently under investigation for Pompe disease, including gene therapy. This review gives an overview of the available knockout mouse models, of the in vitro and in vivo studies performed using recombinant produced enzyme. Furthermore, it describes current therapeutic approaches for Pompe disease as well as experimental therapies like gene correction therapy.

The histone deacetylase inhibitor valproic acid potently augments gemtuzumab ozogamicin-induced apoptosis in acute myeloid leukemic cells.

Gemtuzumab ozogamicin (GO) is a calicheamicin-conjugated antibody directed against CD33, an antigen highly expressed on acute myeloid leukemic (AML) cells. CD33-specific binding triggers internalization of GO and subsequent hydrolytic release of calicheamicin. Calicheamicin then translocates to the nucleus, intercalates in the DNA structure and subsequently induces double-strand DNA breaks. GO is part of clinical practice for AML, but is frequently associated with severe side effects. Therefore, combination of GO with other therapeutics is warranted to reduce toxicity, while maximizing therapeutic selectivity. We hypothesized that the histone deacetylase inhibitor valproic acid (VPA) sensitizes AML cells to GO. VPA-induced histone hyperacetylation opens the chromatin structure, whereby the DNA intercalation of calicheamicin should be augmented. We found that clinically relevant concentrations of VPA potently augmented the tumoricidal activity of GO towards AML cell lines and primary AML blasts. Moreover, VPA treatment indeed augmented the DNA intercalation of calicheamicin and enhanced DNA degradation. Importantly, synergy was restricted to CD33-positive AML cells and did not require caspase activation. In conclusion, the synergistic proapoptotic activity of cotreatment of AML cells with VPA and GO indicates the potential value of this strategy for AML.

Chemokine scavenging by the human cytomegalovirus chemokine decoy receptor US28 does not inhibit monocyte adherence to activated endothelium.

The human cytomegalovirus has found smart ways to exploit the chemokine network in order to subvert immune attack. Chemokines trigger the arrest and firm adhesion of inflammatory cells to the vascular wall. Scavenging of chemokines by viral decoy receptors, such as US28, might prevent arrest of leukocytes to the vascular wall and impair an antiviral immune response. We determined the effect of chemokine scavenging by endothelium-expressed signaling mute US28 (US28R129A) on static monocyte adhesion. Despite the chemokine scavenging capacity of US28R129A, expression of this construct by endothelial cells was insufficient to disrupt leukocyte adhesion to cytokine-activated monolayers. Our results suggest that the concentrations of chemokines that trigger firm leukocyte adhesion are too high to be efficiently scavenged by viral chemokine decoy receptors like US28. From the results of this experimental model a role for US28 in viral immune evasion by chemokine scavenging would appear therefore unlikely.

Closed system generation of dendritic cells from a single blood volume for clinical application in immunotherapy.

Dendritic cells (DC) used for clinical trials should be processed on a large scale conforming to current good manufacturing practice (cGMP) guidelines. The aim of this study was to develop a protocol for clinical grade generation of immature DC in a closed-system. Aphereses were performed with the Cobe Spectra continuous flow cell separator and material was derived from one volume of blood processed. Optimisation of a 3-phase collection autoPBSC technique significantly improved the quality of the initial mononuclear cell (MNC) product. Monocytes were then enriched from MNC by immunomagnetic depletion of CD19+ B cells and CD2+ T cells and partial depletion of NK cells using the Isolex 300I Magnetic cell selector. The quality of the initial mononuclear cell product was found to determine the outcome of monocyte enrichment. Enriched monocytes were cultured in Opticyte gas-permeable containers using CellGro serum-free medium supplemented with GM-CSF and IL-4 to generate immature DC. A seeding concentration of 1 x 10(6) was found optimal in terms of DC phenotype expression, monocyte percentage in culture, and cell viability. The differentiation pattern favours day 7 for harvest of immature DC. DC recovery, viability, as well as phenotype expression after cryopreservation of immature DC was considered in this study. DC were induced to maturation and evaluated in FACS analysis for phenotype expression and proliferation assays. Mature DC were able to generate an allogeneic T-cell response as well as an anti-CMV response as detected by proliferation assays. These data indicate that the described large-scale GMP-compatible system results in the generation of stable DC derived from one volume of blood processed, which are qualitatively and quantitatively sufficient for clinical application in immunotherapeutic protocols.

US28 actions in HCMV infection: lessons from a versatile hijacker.

Mimicking host proteins is a strategy adopted by several herpesviruses to exploit the host cell for their own benefit. In this respect the human cytomegalovirus (HCMV) chemokine receptor homologue US28, has been extensively studied. Molecular pirates such as US28 can teach us about crucial events in HCMV infection and may either offer a potential target for antiviral therapy or provide an alternative strategy to immune suppression. Despite elaborate research into the chemokine binding affinity, signalling properties, intracellular trafficking and expression kinetics of US28, a solid hypothesis about the role of US28 in HCMV infection has not yet been proposed. It appears that US28 may behave as a molecular pirate that employs smart strategies for cell entry, host gene regulation and immune evasion. This review will elaborate on these aspects of US28 biology and discuss possible implications for HCMV infection.

Expression of P2 receptors at sites of chronic inflammation.

Extracellular nucleotides have been identified as important signaling molecules. These nucleotides act on the P2 family of receptors that respond by either forming an ion-channel or by activation of a signal transduction cascade, both of which enable a cellular response. Although a role for P2 receptors in inflammation has been implied, the local expression pattern and kinetics of these receptors at sites of inflammation are not known. Therefore, we have studied the expression of the P2 receptors expressed by inflammatory cells or by cells in the vasculature, with special attention of P2X(1), P2X(7)R, P2Y(1)R, and P2Y(2)R. As a suitable model for studying inflammatory reactions, we have employed the foreign body reaction (FBR), a sterile inflammatory reaction induced by implanting degradable cross-linked dermal sheep collagen disks subcutaneously in the rat. We show that, in the vasculature, the express of P2X(7)R, P2Y(1)R and P2Y(2)R increase until day 2. The expression of P2X(7)R and P2Y(1)R on macrophages and giant cells increased during the course of the inflammatory reaction which was studied for 21 days. The expression of the P2Y(2)R on macrophages and giant cells inside the foreign body increases with time, whereas the expression on macrophages in the surrounding tissue is maximal at day 5. The expression of P2X(1)R remains at a constant low level. The upregulation of P2X(7)R, P2Y(1)R, and P2Y(2)R over time suggests a regulatory function for these receptors in inflammation.

An in vitro model for purging of tumour cells from ovarian tissue.

BACKGROUND: Cryopreservation and autografting of ovarian tissue may preserve fertility after cancer treatment, but could be hampered by tumour cell contamination. Epithelial tumour cell lysis can be obtained with cytotoxic T cell retargeting through the bispecific antibody BIS-1, with combined affinity for the T-cell receptor and epithelial glycoprotein-2 (EGP-2). Our aim was to study the concept of tumour cell purging in the setting of a suspension of ovarian tissue. METHODS: Human ovarian tissue was brought into suspension by mechanical and enzymatic treatment. Cells of the MCF-7 breast cancer cell line and activated human lymphocytes were co- incubated for 4 h with or without BIS-1 and with or without ovarian suspension. After incubation, MCF-7 cell death and cell growth were evaluated by fluorescent cell detection and MTT assay, respectively. Ovarian tissue morphology was evaluated immunohistochemically. RESULTS: MCF-7 cell death and cell growth inhibition increased with increasing ratios of lymphocytes to MCF-7 cells. BIS-1 addition gave further augmentation, with a maximum depletion of growing MCF-7 cells of 89% (SD 11%) versus without BIS-1, 23% (SD 15%; P < 0.001). Follicles remained morphologically intact. CONCLUSIONS: Purging of added epithelial tumour cells from ovarian tissue with BIS-1 is possible in vitro. Morphologically, follicles remain intact after this procedure. This method may contribute to safer replacement of ovarian tissue in female cancer survivors.

A hyaluronan-based nerve guide: in vitro cytotoxicity, subcutaneous tissue reactions, and degradation in the rat.

We investigated possible cytotoxic effects, biocompatibility, and degradation of a hyaluronan-based conduit for peripheral nerve repair. We subjected the conduits to an in vitro fibroblast cytotoxicity test and concluded that the conduits were not cytotoxic. Subsequently, we implanted the conduits subcutaneously in rats, in order to investigate tissue reactions and biodegradation. Initially, a fibrin matrix was formed around the material, while the surroundings were relatively quiet. Macrophages (MØ) migrated to the conduits and formed giant cells next to the material after 5 days. The maximum presence of MØ was found after 3-6 weeks. The appearance of MHC class II cells showed a similar pattern. Highest numbers of giants reached a maximum after 6-12 weeks. Angiogenesis was started in the surroundings of the hyaluronan-based conduit within a few days. Massive ingrowth of blood vessels into the biomaterial was found after 6 weeks as well as cellular ingrowth into the lumen of the tube. At that time the tubular structure of the conduit was lost and loose biomaterial fibers were observed. The results show that a hyaluronan-based conduit is not cytotoxic and shows good biocompatibility. Such a conduit may be suitable as a guide in peripheral nerve repair.

Total white blood cell counts and LPS-induced TNF alpha production by monocytes of pregnant, pseudopregnant and cyclic rats.

Pregnancy in the rat may be associated with an activated innate immune system. Therefore, we investigated monocyte function as well as total white blood cell (WBC) counts during the follicular phase of the ovarian cycle, pregnancy and pseudopregnancy in the rat. Rats were equipped with a permanent jugular vein cannula, and 0.43 ml blood samples were taken from this cannula during the 4 days of the regular oestrus cycle of the rat (n=12). Thereafter, six rats were rendered pregnant, and the other six rats were rendered pseudopregnant according to standard methods. Blood samples were withdrawn from the cannula on days 4, 7 and 11 of pseudopregnancy and on days 4, 7, 11 and 20 of pregnancy. From each blood sample, 0.4 ml was stimulated with lipopolysaccharide (LPS) and monocyte intracellular cytokine production measured using flow cytometry. 30 microl of the blood was used to measure WBC counts and differential WBC counts. The results showed that the number of WBC was significantly increased only on day 11 of pregnancy compared with the follicular phase, and that this was due to the increased numbers of polymorphonuclear (PMN) cells. The percentage of TNF alpha-producing monocytes was increased on all days of pseudopregnancy and on day 11 of pregnancy. The fact that the percentage of monocytes producing TNF alpha upon an LPS stimulus was increased during the post-implantation phase of pregnancy and during pseudopregnancy as compared to the follicular phase may indicate that these conditions are proinflammatory conditions. For the post-implantation phase of pregnancy, this is once more stressed by the increased numbers of WBC and PMN.

Association between macrophage activation and function of micro-encapsulated rat islets.

AIMS/HYPOTHESIS: Survival of microencapsulated islet grafts is limited, even when inflammatory reactions against the capsules are restricted to a small portion of less than 10%. METHODS: This study investigates both in vivo in rat recipients and in vitro whether cellular overgrowth on this minority of the capsules contributes to limitations in the functional survival of the 90% of the encapsulated islets which remain free of any cellular overgrowth. RESULTS: In successful rat recipients of an allogenic microencapsulated islet graft we found that the vast majority of cells in the capsular overgrowth were activated ED-1 and ED-2 positive macrophages which were found in numbers of approximately 1500 per capsule. Co-culture of encapsulated islets with 1500 (nr8383) rat-macrophages per capsule showed that the activation of macrophages was caused by islet-derived bioactive factors since TNF-alpha and IL-1beta secretion by macrophages was induced by islet-containing capsules and not by empty capsules. This activation of macrophages was associated with a decrease in function of the encapsulated islets as evidenced by a quantitatively reduced (35%) insulin response in static incubation and a slower response in perifusion. CONCLUSION/INTERPRETATION: Present research aims to design strategies for the temporary inhibition of macrophage activation since macrophages are predominantly present in the first two months after implantation. These strategies will serve as a pertinent basis for future clinical application of microencapsulated islets.

Cardiac tissue engineering: characteristics of in unison contracting two- and three-dimensional neonatal rat ventricle cell (co)-cultures.

Patients with heart failure have, in spite of improved palliative therapies, bad prognosis. Cardiac tissue engineering by use of a temporary bioscaffold and cardiomyocytes may help to find answers for future treatments in heart failure. For that purpose two neonatal rat heart ventricular cell fractions were obtained after a gradient cell separation. Time related characteristics of Fractions I and II were established in two-dimensional (2-D) and three-dimensional (3-D) cell cultures. The 3-D cardiac constructs were obtained by use of a bovine type I collagen matrix after culturing either under static conditions or in the HARV bioreactor. With the 2-D cultures contracting cells were present after 1 day, and reached confluency from day 5 on and this was maintained up to 135 days. In Fraction-I some non-contracting cells were always noticed between the (in time in unison) contracting cells. Transmission electron microscopy (TEM) revealed that these mainly concerned fibroblasts. Differences in the expression of alpha-SM-1 actin and troponin-T were observed between the two fractions. In both fractions endothelial cells and macrophages were only sporadically observed. All through the 3-D matrix pendant-like single cell and clustered cell contractions were present after 1-2 days, resulting in time in unison contracting of cells with the collagen matrices. The whole event was faster with Fraction-I and was observed up to 3 weeks. At this time point clusters of troponin-T positive cells were found scattered through the collagen matrices. Additionally, TEM revealed healthy layers of connected cardiomyocytes with intercalated discs, in this case on and in between the collagen fibres. These findings provide evidence that in unison contracting structurally organized cell-matrix cardiac constructs can be engineered by use of co-cultures (neonatal cardiomyocytes and fibroblasts) and collagen matrices, which is very promising for the repair of larger scar areas of the myocardium.

Dendritic cells present an intracellular viral antigen derived from apoptotic cells and induce a T-cell response.

We investigated if dendritic cells (DCs) were able to present intracellularly located antigens derived from apoptotic cells to T cells, thereby inducing a CD4+ and a CD8+ response. A transfected cell line with the cytomegalovirus-derived protein pp65 was triggered to go into apoptosis by ultraviolet B (UVB) irradiation, and after the uptake of apoptotic cells by DC, the activation and proliferation of T cells were determined. We found that DC efficiently phagocytosed apoptotic cells and induced a CD4+ and a CD8+ T-cell response specific for the viral protein pp65. This mechanism can be useful for vaccination studies to induce an antiviral immune response.

A possible role for spontaneous interleukin-8 production by acute myeloid leukemic cells in angiogenesis related processes: work in progress.

BACKGROUND: Recently, the role of inter-leukin-8 (IL-8) in angiogenesis was reported. We consequently addressed here the question whether IL-8 produced by acute myeloid leukemia (AML) blasts might have a comparable function. PROCEDURE: In 21 pediatric patients with AML the role of AML derived IL-8 in angiogenesis related processes were investigated. Therefore, IL-8 protein and mRNA expression were measured and endothelial cell (EC) migration and proliferation assays were performed. In addition, bFGF and VEGF mRNA expression were measured by RT-PCR. RESULTS: In the supernatant of the AML blasts, IL-8 protein was present in a varying amount (median 0.86 microg/L, range: 0.1-320 microg/L) and confirmed by RT-PCR. Normal bone marrow mononuclear cells secreted a significant lower amount of IL-8 protein (median: 0.053 microg/L, range: 0.023-0.055 microg/L, P = 0.007). Seven of the 17 tested AML supernatants induced a varying low amount of EC proliferation compared to control media, which was not inhibited by anti-IL-8 antibodies. In contrast, in the EC migration assay, 15 out of the 17 AML supernatants tested, showed an increased EC migration (median fold increase: 1.97, range: 0.66-6.36, P = 0.002) compared to control medium. The increase in EC migration could partially be blocked by anti-IL-8 in 59% of the cases (18% decrease, range 0-62%, P = 0.003). Other contributors for the increase in EC migration were also determined. Vascular endothelial growth factor (VEGF) transcripts by RT-PCR were demonstrated in six out of the nine tested AML cases, while no transcripts for basic fibroblast growth factor (VEGF) could be shown. CONCLUSIONS: Neutralizing anti IL-8 antibodies inhibit EC migration when stimulated with AML supernatant. This suggests a facilitating role for AML-derived IL-8 in an important step in angiogenesis.

Cancer immunotherapy: insights from transgenic animal models.

A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.

The immune response during the luteal phase of the ovarian cycle: increasing sensitivity of human monocytes to endotoxin.

OBJECTIVE: To test the hypothesis that during the luteal phase of the human ovarian cycle, as compared with the follicular phase, the percentage of cytokines producing peripheral monocytes after in vitro stimulation with endotoxin is increased. DESIGN: Prospective study. SETTING: Academic research institution. PATIENT(S): Women with regular menstrual cycles. INTERVENTION(S): Blood samples were collected between days 6 and 9 of the menstrual cycle (follicular phase) and between days 6 and 9 of the menstrual cycle following the LH surge (luteal phase). MAIN OUTCOME MEASURE(S): Percentages of tumor necrosis factor (TNF)-alpha-, interleukin (IL)-1 beta-, and IL-12-producing monocytes as well as total white blood cell (WBC) count, differential WBC counts, and plasma 17 beta-estradiol and progesterone concentrations. RESULT(S): Mean plasma 17 beta-estradiol and progesterone concentrations, percentage of TNF-alpha- and IL-1 beta-producing monocytes, WBC counts, and granulocyte cell count were significantly increased in the luteal phase as compared with the follicular phase of the ovarian cycle. The percentage of IL-12-producing monocytes, monocyte count and lymphocyte count did not vary between the 2 phases of the ovarian cycle. CONCLUSION(S): Together with an increase in progesterone and 17 beta-estradiol during the luteal phase, there is an increase in percentage TNF-alpha- and IL-1 beta-producing peripheral monocytes after in vitro stimulation with endotoxin as compared with the follicular phase of the ovarian cycle. Whether this increased sensitivity of monocytes for proinflammatory stimuli during the luteal phase is due to increased plasma levels of progesterone or 17 beta-estradiol needs further investigation.

Immunohistopathological appearance of three different types of injury in human skin.

OBJECTIVE AND DESIGN: To determine whether underlying mechanisms of inflammation, like cellular infiltrates, expression of adhesion molecule and cytokine patterns are similar under different conditions of injury. Skin biopsies were taken of three different groups of patients in which local inflammation of the skin might occur. MATERIAL, PATIENTS: Skin activation was studied as a result of incision during surgery under aseptic conditions, as a result of a local bacterial infection and as a result of blunt trauma, resulting in a femoral fracture, without disruption of the epithelial barrier. METHODS: Skin biopsies were snap frozen for immunohistochemical analysis. RESULTS: Incision of the skin resulted in a granulocyte infiltrate, paralleled by E-selectin expression. As a result of infection granulocytes were observed and monocyte/macrophage and T cell numbers were increased. Furthermore, E-selectin, VCAM-1 and ICAM-1 expression increased and cytokine expression markedly changed compared to normal skin. Skin taken at the site of the femoral fracture showed no signs of inflammation. CONCLUSION: Different stimuli lead to different local inflammatory responses in human skin.

Susceptibility to development of Mycobacterium ulcerans disease: review of possible risk factors.

Mycobacterium ulcerans disease, also known as Buruli ulcer (BU), is a disease of subcutaneous fat tissue. BU is prevalent in riverine and swamp areas of the tropical zone in Africa, Asia and South America, and a few scattered foci in Australia. The mode of transmission of M. ulcerans has not been fully elucidated, but inoculation into the subcutaneous tissues probably occurs through penetrating skin trauma. BU has not been linked with HIV infection. Antimycobacterial drug treatment is ineffective, and treatment is surgical. Patients eventually develop scars and contractures, with resulting disabilities, and the disease imposes a large burden on affected populations. The incidence of BU has dramatically increased in West African countries over the last decade. There is an urgent need for research into host and environmental risk factors for BU in order to develop effective strategies to combat this disease. We review possible genetic host susceptibility factors for BU that are relevant in other mycobacterial diseases: natural resistance-associated macrophage protein-1 (NRAMP-1), HLA-DR, vitamin D3 receptor, mannose binding protein, interferon-gamma (IFN-gamma) receptor, tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1 alpha, 1 beta and their receptor antagonists; and IL-12. Schistosoma haematobium infection is highly endemic in many BU foci in West Africa, with a striking increase in transmission after river dams were constructed. This observation, and the observations from interaction of schistosomiasis and tuberculosis, have fueled our hypothesis that schistosomiasis is a risk factor for BU by driving the host immune response towards a predominantly Th-2 pattern, away from a Th-1 preponderant protection against mycobacterial infection. If the latter hypothesis is confirmed, enhanced schistosomiasis control should impact on BU.

Mephenytoin as a probe for CYP2C19 phenotyping:effect of sample storage, intra-individual reproducibility and occurrence of adverse events.

AIMS: To further evaluate mephenytoin as a probe for CYP2C19 phenotyping. METHODS: Healthy subjects (n = 2638) were phenotyped using the urinary (S)-mephenytoin to (R)-mephenytoin ratio. This method was evaluated for (a) the stability of the S/R-ratio following sample storage, (b) the intraindividual reproducibility of the ratio, and (c) the occurrence of adverse events. RESULTS: After prolonged storage, the S/R-ratio of samples from extensive metabolisers (EM) increased up to 85%. In 1.5% of the cases (1 out 66), this led to incorrect classification of phenotype. In EMs, but not in poor metabolisers (PMs), the S/R-ratio increased after acid treatment. The intraindividual reproducibility of the mephenytoin phenotyping procedure was 28%. No major side-effects were observed and there was no relationship between the incidence of side-effects and the phenotype of the subject. CONCLUSIONS: After prolonged storage the S/R-ratio significantly increased in EMs and, although low, the risk of incorrect classification should not be ignored. Our data support the use of mephenytoin as a safe drug for CYP2C19 phenotyping.

Tumor necrosis factor alpha (TNFalpha) in human skin: a comparison of different antibodies for immunohistochemistry.

Conflicting results have been reported regarding the localization and presence of TNFalpha in normal human skin. To study TNFalpha expression, we tested a panel of antibodies directed against human TNFalpha. First, antibodies were tested for immunoreactivity on cytospots of isolated LPS-stimulated peripheral blood mononuclear cells. Second, antibodies were tested to detect recombinant TNFalpha in Western blots. Some antibodies were found to be unable to detect recombinant TNFalpha in the blots. However, most antibodies were able to bind TNFalpha protein, but they did not bind to other irrelevant proteins that were also present in the blots. Finally, antibodies were tested on cryosections of normal human skin. Antibodies that did not react with TNFalpha in the blots were incubated with TNFalpha before the staining procedure to see whether these antibodies specifically bound TNFalpha. We found that, although all the antibodies bound TNFalpha, there were clear differences in staining patterns. This indicates that these antibodies may recognize distinct epitopes or different forms of TNFalpha. The differences found in this study and those reported previously could be the result of differences in the concentration of antibody used, the staining procedure or specificity of the antibody itself. So, for unambiguous interpretation of data, it is important to know the characteristics of the antibodies used.