[Tubercular osteomyelitis of the clivus and the nasopharynx]. / Tuberkulöse Osteomyelitis des Klivus mit Beteiligung des Nasopharynx.

Involvement of the skull base is rare in tuberculosis. We report here the case of a 28-year-old female patient with an osteolytic process of the clivus with compression of the brain stem and involvement of the nasopharynx. She reported suffering from headaches for the last 6 months, and diplopia had occurred 1 week before her diagnosis as a result of paresis of the VIth cranial nerve on the right side. A biopsy was obtained endoscopically via a transnasal approach, revealing a granulomatous inflammation with acid-fast rods and thus confirming the diagnosis of a tuberculoma. When the biopsy was taken there was no evidence of any further tuberculomas in this patient. The clinical picture, diagnosis, and treatment of tuberculosis of the skull base and nasopharynx are discussed and the literature on this rare clinical entity is reviewed with reference to this patient's case report.

Ethanol decreases expression of p21 and increases hyperphosphorylated pRb in cell lines of squamous cell carcinomas of the head and neck.

BACKGROUND: Alcohol increases the risk of cancers of the upper aerodigestive tract, but the biological mechanisms of this ethanol effect are still unclear. We recently reported that ethanol is able to induce in vitro proliferation accompanied by an increased number of cells in the S phase of the cell cycle in squamous cell carcinoma cell lines of the head and neck (SCCHN). In the current study we investigated the influence of ethanol over a limited period of time (96 hr) on cell cycle-regulating proteins involved in G1/S phase transition. METHODS: Synchronized cells of SCCHN cell lines JPPA (larynx) and SCC 9 and SCC 25 (tongue), as well as HaCaT (human immortalized keratinocytes)-used as a control-were cultured for 96 hr in the presence or absence of ethanol (10-3M). At several time intervals the expression of cyclin D1 and p21 and the phosphorylation status of the retinoblastoma protein (pRb) were determined by Western or Northern Blot analysis, or both. RESULTS: Ethanol had no influence on the protein expression of cyclin D1. In contrast, a distinct downregulation of p21 at the protein as well as the mRNA level could be detected. Furthermore, as a downstream event, the hyperphosphorylated form of the pRb increased. CONCLUSIONS: In the acute alcohol in vitro experiments, the marked downregulation of the important cell cycle inhibitor p21 and the corresponding increase of hyperphosphorylated pRb accelerate the progression of cells from the G1 to the S phase in the cell cycle. The importance of these data and their relevance to in vivo conditions remain speculative, but it could be a critical step in the multistep process of SCCHN carcinogenesis induced by ethanol.

1,25(OH)2 vitamin D3 induces elevated expression of the cell cycle-regulating genes P21 and P27 in squamous carcinoma cell lines of the head and neck.

The biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], inhibits proliferation and induces differentiation for various malignant cells, including squamous cell carcinoma cell lines of the head and neck (SCCHN). These effects are due to an arrest of cells in the G0/G1 phase of the cell cycle and are predominantly mediated by the vitamin D receptor. To further explore the molecular mechanisms of the antiproliferative activity in SCCHN we studied the influence of 1,25(OH)2D3 on the expression of the G1 phase-regulating proteins cyclin D1, p21 and p27. Furthermore, as a direct target of G1 protein complexes, we investigated the phosphorylation status of the retinoblastoma protein (pRb). Synchronized cells of 2 SCCHN cell lines [JPPA (laryngeal carcinoma) and SCC 9 (tongue carcinoma)] and human immortalized keratinocytes (HaCaT) were cultured for 96 h in the presence or absence (ethanol as control) of 1,25(OH)2D3 (10(-7) M). At various time intervals the cell cycle status was detected by fluorescence-activated cell sorting (FACS) analysis and in parallel the expression of cell cycle-regulating proteins was determined at the protein and mRNA levels. In all cell lines tested 1,25(OH)2D3 caused an arrest of cells in the G0/G1 phase of the cell cycle and markedly induced the expression of the inhibitors p21 and p27. No influence was detectable on the expression of cyclin D1. Induction of p21 and p27 mRNA revealed transcriptional regulation by the vitamin D receptor. Simultaneously, hyperphosphorylated pRb was transformed to the hypophosphorylated form. Our results demonstrate that the biologically active form of vitamin D3 directly regulates the expression of p21 and p27, inducing a G0/G1 phase arrest: one mechanism by which 1,25(OH)2D3 controls cell proliferation inSCCHN.

Non-steroidal anti-inflammatory drugs inhibit telomerase activity in head and neck squamous carcinoma cell lines.

BACKGROUND: Antiproliferative effects in neoplastic cells of different origin have been attributed to non-steroidal anti-inflammatory Drugs (NSAIDs) during the past few decades. METHODS: We tested the influence of NSAIDs and hydrocortisone on cell lines derived from head and neck squamous cell cancer (HNSCC) and on normal oral mucosal keratinocytes. Cell numbers were assayed by cell counting, proliferation, telomerase activity with a colorimetric assay, and cell cycle distribution by flow cytometry. RESULTS: In the neoplastic cell lines indomethacin and ibuprofen caused a dose-dependent reduction of cell numbers and telomerase activity without altering cell viability and increased the percentage of cells in G0/G1 phase. In normal oral mucosal keratinocytes, only minor effects could be detected in response to NSAIDs and hydrocortisone. CONCLUSION: These results demonstrate that NSAIDs have activity against HNSCC cells in vitro and may have clinical applications in combination with other therapeutic regimens.

Non-steroidal anti-inflammatory drugs induce apoptosis in head and neck cancer cell lines.

Non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit tumorigenesis in colorectal cancer due to the induction of apoptosis. Disturbances of cellular pathways ultimately leading to apoptosis may contribute to the process of neoplastic transformation and immortalization. In this study we wanted to determine the influence of different NSAIDs (indomethacin, ibuprofen and sodium salicylate) and hydrocortisone on Bcl-2 expression and the apoptotic behavior of head and neck tumor cell lines and normal oral keratinocytes. Bcl-2 expression was determined by monoclonal antibody staining and fluorescence-activated cell-sorting measurement. Apoptotic cells were visualized with a epifluorescence microscope after staining with CytoDeath M30 antibody. Indomethacin (1 mM) and ibuprofen (1 mM) significantly reduced Bcl-2 expression in the cancer cell lines tested and might be thought responsible for the observed increase in apoptosis. At all concentrations tested the influence of sodium salicylate and hydrocortisone on Bcl-2 expression was not significant. In contrast, the NSAIDs tested had only a minor influence on normal oral keratinocytes. Our results demonstrate a significant reduction in growth and an increase in apoptosis, possibly due to a reduction in Bcl-2 expression. after exposure to indomethacin and ibuprofen in the head and neck cancer cell lines tested.

The role of fibroblasts from oropharyngeal mucosa in producing proinflammatory and mitogenic cytokines without prior stimulation.

Cytokine production by fibroblasts is not only important for immunological and inflammatory reactions in the epidermis and mucosa, but also for growth and differentiation of epithelial cells. To characterize the role of fibroblasts in the oropharyngeal mucosa, the expression of a panel of cytokines and cytokine receptors by fibroblasts isolated from normal human oropharyngeal mucosa was investigated by enzyme-linked immunosorbent assay (ELISA), reverse transcribed polymerase chain reaction (RT-PCR) and flow cytometry (FACS). Oropharyngeal fibroblasts produced the proinflammatory cytokines interleukin 1 alpha (IL-1 alpha), IL-6 and IL-8 without addition of phorbol-12-myristate-13-acetate (PMA) or biological response modifiers, suggesting an active involvement of these cells in host defence mechanisms. Keratinocyte growth factor (KGF), a growth factor for epithelial cells, and the angiogenetic fibroblast growth factors acidic and basic FGF (aFGF, bFGF) were also synthesized. Expression of receptors for IL-1, IL-4, IL-6, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) was found. These results indicate that oral fibroblasts are capable of producing a number of cytokines without the need for additional stimuli and emphasize their active regulatory role in the maintenance of the oral mucosa.

Effects of ethanol treatment of proliferation and differentiation in a head and neck squamous cell carcinoma cell line.

INTRODUCTION: Epidemiologic studies have provided evidence that chronic ethanol consumption is an independent risk factor in upper aerodigestive tract cancer, but the underlying mechanisms are largely unknown. METHODS: To examine ethanol effects on mucosal keratinocytes in vitro, we used a squamous cell carcinoma of the head and neck (SCCHN) cell line as a model and, to exclude line specific effects, two other cell lines. The influence of ethanol on proliferation (using [3H]thymidine uptake/cell number), cell cycle distribution, cytokeratin pattern, and growth factor receptor expression (using FACS analyses) was investigated. RESULTS: Ethanol increased in a dose dependent manner (tested range 10(-3) M to 10(-10) M) the [3H]thymidine uptake and cell number, with unaltered viability (>95%) in all concentrations. In all tested cell lines, addition of 10(-3) M ethanol caused: (a) a significant increase of cells in the S-phase of the cell cycle; (b) a shift of cytokeratin pattern that suggested inhibition of differentiation; and (c) significant upregulation of EGF, IL-4, and PDGF receptors. CONCLUSIONS: Our results demonstrated an increased proliferation and reduced differentiation induced by ethanol in mucosa derived neoplastic cells, which may enhance further growth of neoplastic cells. These effects may also be involved in the carcinogenesis of upper aerodigestive tract malignancies.

Cytokine expression of human oral keratinocytes.

The present study aimed to examine the cytokine expression pattern of human oral mucosa-derived keratinocytes at the protein and RNA level. The mRNA expression was measured by a RT-PCR method and the protein production was determined by an ELISA technique. In freshly isolated oral keratinocytes, IL-1alpha (interleukin 1alpha), IL-6, IL-8, transforming growth factor beta, tumor necrosis factor alpha and basic fibroblast growth factor were detectable at the protein and mRNA level, whereas platelet-derived growth factor, acidic fibroblast growth factor and transforming growth factor alpha were found only at the mRNA level. There were no detectable signals of IL-2 and IL-4. The cytokine production at the protein level was independent of prior stimulation with phorbol myristate acetate. Our results show that human oral keratinocytes - under nonpathological conditions - produce a cytokine pattern quite similar to that of epidermal keratinocytes, which may have implications for further functional studies.

Non-radioactive semiquantitative testing for the expression levels of telomerase activity in head and neck squamous cell carcinomas may be indicative for biological tumour behaviour.

Head and neck cancer arises and progresses through specific genetic alterations which lead to an invasive immortal phenotype. The process of immortalization is associated with the activation of the enzyme telomerase, a ribonucleoprotein with reverse transcriptase activity which is capable of synthesizing telomeric repeats at the end of chromosomes. This enzyme is expressed in nearly all neoplasms and germline cells and is absent in most normal human somatic cells. Because of this expression pattern testing for telomerase activity may deliver useful diagnostic and/or prognostic information about clinical tumour behaviour. Telomerase activity was therefore analysed in 16 primary lesions of head and neck squamous cell carcinomas (HNSCC) using the polymerase chain reaction-based telomeric repeat amplification protocol (TRAP). For a sensitive semiquantitative analysis of telomerase activity TRAP products were mixed with Pico Green I and the fluorescence emission intensities were measured. All 16 samples tested positive. When the Pico Green I data were compared with clinical parameters, it was obvious that N0 necks revealed significantly (p < 0.05) lower emission intensities (i.e. telomerase activity) than N + necks. Our results indicate that a high telomerase activity in HNSCC may facilitate lymph node metastasis and that the estimation of telomerase activity is a useful diagnostic tool which could influence treatment modalities.

Different KGF expression in squamous cell carcinomas of the head and neck and in normal mucosa.

Fibroblast growth factors (FGFs) are major factors mediating epithelial-mesenchymal interactions in the epidermis and mucosa. In this study the expression of the FGFs keratinocyte growth factor (KGF), acidic FGF (aFGF) and basic FGF (bFGF) was examined in tumour tissue specimens from 14 patients with advanced-stage squamous cell carcinoma of the head and neck (SCCHN) and 3 SCC cell lines by reverse-transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry. None of the SCC cell lines was positive for KGF mRNA, whereas all cell lines were highly positive for aFGF and bFGF. In SCCHN tissue samples the level of KGF mRNA expression was significantly lower than in normal mucosa. Tumour stroma and the submucosal areas of normal mucosa stained intensely with anti-KGF antibody in immunohistochemical slides, whereas tumour cell nests were negative. Exposure of SCC cells to KGF thus differs from normal mucosa both quantitatively and regarding spatial distribution. This fact and the overexpression of aFGF and bFGF by tumour cells potentially promote tumour growth, invasion and metastasis. Since these growth factors and their receptors are well characterized, these observations could lead to new therapeutic strategies in SCCHN, for instance by blocking their receptors or antisense targeting.

Oral keratinocytes derived from the peritonsillar mucosa express the proinflammatory cytokine IL-6 without prior stimulation.

The aim of the present study is to examine the cytokine expression and corresponding receptor pattern of human oral mucosa-derived keratinocytes. The mRNA expression of these cytokines from isolated and purified cells was measured by a RT-PCR method, the protein production by ELISA, and the receptor expression was determined by FACS analysis. In freshly isolated oral keratinocytes, IL-1alpha, IL-1alpha receptor antagonist, IL-6, IL-8, TGF-beta, TNF-alpha, and bFGF were detectable at the protein and mRNA level, whereas PDGF and TGF-alpha were found only at the mRNA level. There were no detectable signals for IL-2 and IL-4. The cytokine production at the protein level was independent from stimulation with PMA (phorbol myristate acetate). Unstimulated commercially available and primary isolated epidermal keratinocytes showed similar cytokine pattern except a lack of IL-6. FACS analysis revealed receptor expression on oral keratinocytes for IL-1, IL-2, IL-4, IL-6, EGF, IFN-gamma, and PDGF. In addition, receptor mRNA for IL-8, TNF-alpha, FGF-2 and KGF could be detected, but not for IL-10. Our results show that human oral keratinocytes produce a cytokine panel comparable to epidermal keratinocytes. In contrast to the epidermis, IL-6 was produced by human oral keratinocytes constitutively without prior stimulation, which may indicate their active regulation role in the maintenance of the oral mucosa.

Magnetic cell separation for purification of human oral keratinocytes: an effective method for functional studies without prior cell subcultivation.

In studying human oral keratinocytes, it would be very helpful to obtain a pure population of cells without prior in vitro expansion. An immunomagnetic separation technique, or magnetic cell separation (MACS), was modified for efficient purification of human oral keratinocytes. Subsequent to two-step enzymatic digestion, the cell suspension was labelled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells. In a second step a rat anti-mouse antibody conjugated with colloidal superparamagnetic particles was used. Labelled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. The unlabelled, unretained cells were further examined by flow cytometry analysis, enzyme-linked immunosorbent assay and polymerase chain reaction. After the MACS procedure, unretained cells showed a strong positivity for the lu-5 MoAb (as a marker for pan-cytokeratin) and were negative for anti-vimentin (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte-detecting antibodies, thus representing pure keratinocytes (> 98%). Purified keratinocytes maintained full viability (> 91%) and functional capacities. [3H]thymidine uptake and epidermal growth factor (EGF) receptor expression were unaltered when compared with the non-separated cell population. Furthermore, interleukin-1 alpha was detected at the protein and RNA levels in keratinocytes immediately after MACS enrichment. Our findings show that MACS appears to be a useful tool for purification of oral keratinocytes and allows for further functional studies without prior subcultivation of cells.

Treatment of a local recurrence of a carcinoid tumor of the middle ear by extended subtotal petrosectomy.

A recurrence of a primary carcinoid tumor of the middle ear 15 years after radical tympanomastoidectomy is reported. An extended subtotal petrosectomy using a craniocervical approach with temporary infracondylar mandibulotomy was performed, since imaging studies demonstrated an extensive tumor with a close relationship to the tegmen tympani, facial nerve, and ascending and horizontal portions of the carotid canal. The tumor was metabolically inactive. Histopathological examination showed a solid, trabecular tumor that was positive for pancytokeratin Lu5, neuron-specific enolase, pancreatic intestinal polypeptide and glucagon. Neuroendocrine-granules were demonstrable under electron microscopy. This case is reported to show that primary middle-ear carcinoid tumors can recur years after radical tympanomastoidectomy.

Myocutaneous flaps in patients with head and neck cancer retain their immunological capacities in an activated functional state.

Free/pedicled myocutaneous flaps used as functional replacement after radical dissection of advanced stage squamous cell carcinomas of the oral cavity/oropharynx were examined by immunohistochemistry (APAAP-technique). Biopsies from eight patients were taken at the time of surgery and at 3 and 5 months post-operatively. Fifteen monoclonal antibodies were used to detect surface antigens as markers of phenotypic changes of immune competent cells. In post-operative biopsies all antigens investigated increased significantly. Significantly higher numbers of CD45RO+ (P < 0.01) and CD45RA+ (P < 0.001) leukocytes were detectable. The majority of these leukocytes were TcR alpha/beta +/CD3+ T-cells, which increased in the CD4 (P < 0.05) and the CD8 (P < 0.001) subset. In addition, B-cells (P < 0.05), granulocytes (P < 0.05), NK cells (CD16+ lymphocytic cells; P < 0.05) and mature macrophages (25F9+cells; P < 0.01) were increased. Intra- and subepidermally a significantly (P < 0.01) higher number of dendritic-/Langerhans cells (CD1a+) was detectable. In post-operative biopsies, the activation-associated antigens ICAM-1, VCAM and HLA-DR were expressed on significantly more mononuclear-/endothelial cells and on keratinocytes. Our findings indicate that the myocutaneous flaps still contained cells with immunological capacities.

IL-1 and TNF-alpha but no IL-2 expression is found in squamous cell carcinomas of the head and neck by RT-PCR.

We studied the expression of IL-1 (Interleukin 1), IL-2, IL-4, IL-10, GM-CSF (granulocyte/macrophage colony stimulating factor), TNF-alpha (tumor necrosis factor alpha) and IFN-gamma (Interferon gamma) in tumor specimens and adjacent mucosa from 12 patients with squamous cell carcinoma of the head and neck (SCCHN) by reverse-transcribed polymerase chain reaction (RT-PCR). No IL-2 and IL-4 expression was detected in any of the tumor specimens. High level expression of IL-1 and TNF-alpha was found in all 12 samples analyzed. Expression of GM-CSF was detected in 7 (58%) of 12 tumors, IL-10 was found in 3 (25%) and IFN-gamma in 6 (50%) of the samples. No qualitative differences in the cyotokine expression pattern were found between normal and tumor tissue. Our data indicate that there is a lack of cytokines typical for a specific, T-cell mediated immune response in SCCHN. A number of cytokines which are required for the initial steps of immune response however are produced. These results might have implications for future immuno-chemotherapeutical strategies.

Antiproliferative effects of the biologically active metabolite of vitamin D3 (1,25 [OH]2 D3) on head and neck squamous cell carcinoma cell lines.

In addition to a role in calcium and phosphate homeostasis other vitamin D receptor (VDR) mediated effects have been discovered during the past few years for the biologically active metabolite of vitamin D, 1,25(OH)2D3. An antiproliferative, differentiation-inducing effect on non-malignant and neoplastic cells of different origin has now been described. We examined the influence of 1,25(OH)2D3 on human squamous cell carcinomas of the head and neck (SCCHN). A differentiated (JP-PA) and undifferentiated (LF-FR) SCCHN line was studied with respect to proliferative capacity (using [3H]-thymidine uptake and cell number) and cell cycle distribution as determined by flow cytometry (FACS). Both cell lines were positive for VDR, which was found to be increased after the addition of 10(-7) M 1,25(OH)2D3, as shown by FACS analyses. The administration of 1,25(OH)2D3 at a concentration between 10(-7) M and 10(-10) M caused a dose-dependent moderate growth inhibition, as reflected by down-regulation of DNA synthesis (reduced [3H]-thymidine uptake) and a decrease in cell numbers. The JP-PA cell line showed a significant growth reduction for both concentrations tested, whereas for LF-FR a significant inhibition was detected only for 10(-7) M. The addition of 10(-7) M 1,25(OH)2D3 caused a blockade in the transition of cells from G1 to S phase in both cell lines, with a significant accumulation of cells in the G0/G1 phase. Our results demonstrate a receptor-mediated, dose-dependent inhibition of neoplastic growth by 1,25(OH)2D3 in human SCCHN lines.

[Direct detection of Toxoplasma gondii with polymerase chain reaction in diagnosis of fetal toxoplasma infection]. / Direkter Nachweis von Toxoplasma gondii mit Polymerase-Kettenreaktion zur Diagnostik einer fetalen Toxoplasma-Infektion.

Primary infection with Toxoplasma gondii during pregnancy may affect the fetus and result in congenital toxoplasmosis. In Austria serological screening for detection of newly acquired infection during pregnancy was introduced in 1975. In this study we used polymerase chain reaction (PCR) for detection of fetal infection with Toxoplasma gondii. Amniotic fluid samples were analyzed from 11 women with serological indication of acute toxoplasmosis infection. Nine of these women had already received treatment prior to amnio-centesis and no evidence of Toxoplasma gondii DNA was detected with PCR in the respective amniotic fluid samples. Isolation of the organism by mouse inoculation was negative in these cases and follow-up serology as well as clinical examination of the infants confirmed these results. In 2 patients investigation of the amniotic fluid samples by means of PCR was positive; both women had not yet been treated at the time of amniocentesis. Our results indicate that identification of Toxoplasma gondii in amniotic fluid is a useful procedure for diagnosing or excluding fetal infection. Moreover, the current recommendations of the screening program appear to be successful in preventing congenital toxoplasmosis.

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.

Altered regulation of apolipoprotein A-IV gene expression in the liver of the genetically obese Zucker rat.

Apolipoprotein (apo) A-IV, a structural component of chylomicrons and high-density lipoproteins, may play a role in the catabolism of triglyceride-rich lipoproteins and in reverse cholesterol transport. To study the regulation of apoA-IV gene expression by genetic and nutritional factors, we determined the effect of a fish oil-rich and a sucrose-rich diet on apoA-IV gene transcription and nuclear and total cellular apoA-IV mRNA abundance in livers of genetically obese, hyperlipoproteinemic (fa/fa) Zucker rats and their lean (Fa/-) littermates. In obese rats fed chow, hepatic apoA-IV gene expression was more than twofold higher than in lean rats because of a post-transcriptional mechanism. apoA-I gene expression and apoC-III mRNA levels, studied as controls, were similar in both groups. The fish oil-rich diet reduced total cellular apoA-IV mRNA abundance transcriptionally to 34 +/- 4% of basal values in lean rats, but did not alter apoA-IV gene expression in obese rats. In contrast, this diet reduced apoA-I gene expression in both lean and obese animals. The sucrose-rich diet increased apoA-IV gene expression twofold in both lean and obese rats. Thus, genetic obesity alters the response of hepatic apoA-IV gene expression to a lipid-lowering diet rich in fish oil by a mechanism affecting transcriptional regulation.

Evaluation of radioimmunoassays: comparison of dose interpolation calculations by four parameter logistic and spline functions.

Two computerized methods for dose interpolation calculation were compared. Generated data sets with a known coefficient of variation as well as laboratory RIA data were analysed. The four parameter logistic method, which is based on an approximation of the mass action law, performed better than the Spline method, a procedure which makes no a priori assumptions about the data. Correct weighting of the data was important for obtaining satisfactory fits. The determination of the response error relationship proved to be the most satisfactory approach in obtaining suitable weighting factors.