Secreted frizzled-related protein 2 (SFRP2) expression promotes lesion proliferation via canonical WNT signaling and indicates lesion borders in extraovarian endometriosis.

STUDY QUESTION: What is the role of SFRP2 in endometriosis? SUMMARY ANSWER: SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis, regulating endometriosis lesion growth and indicating endometriosis lesion borders together with CTNNB1 (also known as beta catenin). WHAT IS KNOWN ALREADY: Endometriosis is a common, chronic disease that affects women of reproductive age, causing pain and infertility, and has significant economic impact on national health systems. Despite extensive research, the pathogenesis of endometriosis is poorly understood, and targeted medical treatments are lacking. WNT signaling is dysregulated in various human diseases, but its role in extraovarian endometriosis has not been fully elucidated. STUDY DESIGN, SIZE, DURATION: We evaluated the significance of WNT signaling, and especially secreted frizzled-related protein 2 (SFRP2), in extraovarian endometriosis, including peritoneal and deep lesions. The study design was based on a cohort of clinical samples collected by laparoscopy or curettage and questionnaire data from healthy controls and endometriosis patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Global gene expression analysis in human endometrium (n = 104) and endometriosis (n = 177) specimens from 47 healthy controls and 103 endometriosis patients was followed by bioinformatics and supportive qPCR analyses. Immunohistochemistry, Western blotting, primary cell culture and siRNA knockdown approaches were used to validate the findings. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 220 WNT signaling and CTNNB1 target genes analysed, 184 genes showed differential expression in extraovarian endometriosis (P < 0.05) compared with endometrium tissue, including SFRP2 and CTNNB1. Menstrual cycle-dependent regulation of WNT genes observed in the endometrium was lost in endometriosis lesions, as shown by hierarchical clustering. Immunohistochemical analysis indicated that SFRP2 and CTNNB1 are novel endometriosis lesion border markers, complementing immunostaining for the known marker CD10 (also known as MME). SFRP2 and CTNNB1 localized similarly in both the epithelium and stroma of extraovarian endometriosis tissue, and interestingly, both also indicated an additional distant lesion border, suggesting that WNT signaling is altered in the endometriosis stroma beyond the primary border indicated by the known marker CD10. SFRP2 expression was positively associated with pain symptoms experienced by patients (P < 0.05), and functional loss of SFRP2 in extraovarian endometriosis primary cell cultures resulted in decreased cell proliferation (P < 0.05) associated with reduced CTNNB1 protein expression (P = 0.05). LIMITATIONS REASONS FOR CAUTION: SFRP2 and CTNNB1 improved extraovarian endometriosis lesion border detection in a relatively small cohort (n = 20), although larger studies with different endometriosis subtypes in variable cycle phases and under hormonal medication are required. WIDER IMPLICATIONS OF THE FINDINGS: The highly expressed SFRP2 and CTNNB1 improve endometriosis lesion border detection, which can have clinical implications for better visualization of endometriosis lesions over CD10. Furthermore, SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis and positively regulates endometriosis lesion growth, suggesting that the WNT pathway may be an important therapeutic target for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Academy of Finland and by Tekes: Finnish Funding Agency for Innovation. The authors have no conflict of interest to declare.

Topical cis-urocanic acid attenuates oedema and erythema in acute and subacute skin inflammation in the mouse.

BACKGROUND: cis-Urocanic acid (cis-UCA) is an endogenous immunosuppressive molecule of the epidermis. OBJECTIVES: We investigated the effects of topical cis-UCA creams (2·5% and 5%) in acute and subacute mouse models of skin inflammation. METHODS: Acute skin irritation was induced by applying dimethyl sulphoxide (DMSO) on the earlobe of CD-1 mice. Topical cis-UCA, hydrocortisone (1%) or tacrolimus (0·1%) were applied 10 min later. In another model, subacute inflammation was provoked and maintained by three applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the ears of NMRI mice on days 1, 2 and 4. The test products were applied topically twice a day during 6 days. RESULTS: In the acute DMSO model, cis-UCA creams suppressed ear swelling at 1 h significantly more efficiently than hydrocortisone (P < 0·01) and tacrolimus (P < 0·001). Ear swelling was significantly inhibited by cis-UCA (P < 0·001) in the subacute TPA model as well. The 5% cream also decreased erythema, whereas tacrolimus enhanced skin reddening. Treatments with cis-UCA did not affect TPA-induced infiltration of neutrophils to the skin. In contrast to hydrocortisone, cis-UCA did not reduce epidermal thickness. CONCLUSIONS: The results suggest that cis-UCA - unlike hydrocortisone and tacrolimus - is efficient in both acute and subacute skin inflammation, attenuating skin oedema and erythema. Topical drug therapy with cis-UCA may provide a safe and effective drug treatment modality in inflammatory skin disorders.

Large performance variation does not affect outcome in the Finnish cervical cancer screening programme.

OBJECTIVE: Cytology screening for prevention of cervical cancer can reduce incidence and mortality by more than 80% in settings with good organization and rigorous quality control. Audit studies are essential for reaching and maintaining a high quality of screening. The aim of this study was to evaluate variation in performance indicators by screening laboratory and assess the impact on the effectiveness of screening as indicated by cervical intraepithelial neoplasia grade 3 and above (CIN3+) rates after a negative screen. METHODS: Seven cytology screening laboratories operating during 1990-1999 with a total of 953 610 screening tests performed were included in the study. By linking screening and cancer register files, all cases of CIN3+ diagnosed in the screened population were identified. For 395 CIN3+ cases with a preceding negative screen and 787 controls, a re-evaluation of smears was undertaken to uncover false negative screening tests. Performance parameters and rates of CIN3+ after a negative screen were analysed for interlaboratory heterogeneity. RESULTS: The rates of follow-up recommendations and referrals varied by up to 3.6- (2.8-10.2%) and 4.0-fold (0.03-0.12%), respectively. CIN1, CIN2 and CIN3+ screen detection rates differed by up to 8.5- (0.02-0.17%), 5.4- (0.05-0.25%) and 3.3-fold (0.05-0.18%). False negative rates determined by re-evaluation showed up to 2.1-fold differences (29-62%). Rates of CIN3+ after a negative screen (0.023-0.048%) and as a proportion of total CIN3+ (15-31%) in the screened population were low and did not vary significantly. CONCLUSIONS: There were large variations in the sensitivity-specificity trade-off between laboratories, reflected in all performance indicators as well as in the test validity estimates of the re-evaluation phase, but not in screening effectiveness. Even though performance variations do not always have an impact on the effectiveness of screening, they lead to variations in cost, treatment and psychological burden, and should be addressed.

Serum HE4 concentration differentiates malignant ovarian tumours from ovarian endometriotic cysts.

Human epididymal secretory protein E4 (HE4, also known as WAP four-disulphide core domain protein 2) is a new promising biomarker for ovarian cancer but its specificity against ovarian endometriotic cysts is only superficially known. We, thus, analysed serum HE4 concentrations together with a tumour marker CA125 in serum samples of women diagnosed with various types of endometriosis, endometrial cancer or ovarian cancer, and in samples from healthy controls. The mean serum concentration of HE4 was significantly higher in serum samples of patients with both endometrial (99.2 pM, P<0.001) and ovarian (1125.4 pM, P<0.001) cancer but not with ovarian endometriomas (46.0 pM) or other types of endometriosis (45.5 pM) as compared with healthy controls (40.5 pM). The serum CA125 concentrations were elevated in patients with ovarian cancer, advanced endometriosis with peritoneal or deep lesions, or ovarian endometriomas, but not in the patients with endometrial cancer. The microarray results revealed that the mRNA expression of the genes encoding HE4 and CA125 reflected the serum protein concentrations. Taken together, measuring both HE4 and CA125 serum concentrations increases the accuracy of ovarian cancer diagnosis and provides valuable information to discriminate ovarian tumours from ovarian endometriotic cysts.

Estrogen receptor-beta is the predominant estrogen receptor subtype in human oral epithelium and salivary glands.

Many studies have shown that the oral mucosa and salivary glands are sensitive to estrogen action. However, the expression of estrogen receptors (ERs) within these tissues is an area of controversy. ERs exist as two subtypes (ERalpha and ERbeta), and we hypothesized that the incongruity between ER expression and estrogen sensitivity may result from differential expression of ER subtypes in oral tissues. To test this hypothesis, we analyzed oral mucosal and salivary gland samples for ERalpha and ERbeta protein expression by immunohistochemistry from a cross-section of patients attending hospital for surgical problems of the head and neck. ERalpha was not detected in oral buccal and gingival epithelium or in salivary glands. In contrast, ERbeta was widely expressed at high levels in all oral tissues studied. Within these tissues, ERbeta was observed primarily in keratinocytes and salivary gland acinar and ductal cells. Our results demonstrating the expression of only the ERbeta subtype within oral tissues may explain the contradictory results from previous studies investigating ER expression in these tissues. Importantly, these results suggest that estrogens may act via ERbeta in oral tissues and explain the effect of hormonal changes on the oral mucosa as well as on saliva secretion and composition.

Apoptotic index and bcl-2 expression as prognostic factors in colorectal carcinoma.

OBJECTIVES: To examine programmed cell death in 57 colorectal carcinomas (49 primary tumours and 8 metastases) and determine the prognostic significance of apoptosis in colorectal cancer. METHODS: Apoptotic index (AI) was ascertained by counting apoptotic bodies, using terminal deoxynucleotidyl transferase mediated digoxigenin nick end labelling (Tunel assay) and the expression of bcl-2 was examined immunohistochemically. Statistical analysis was used to test the value of clinical variables, histopathological data, AI and bcl-2 expression in predicting the clinical outcome of these patients and the survival function was calculated using the Kaplan-Meier method. RESULTS: AI was found to have a significant independent effect on survival (p = 0.0006), with lower values of AI conveying better survival. CONCLUSION: In summary, these findings reveal that AI is a useful prognostic factor in colorectal carcinoma.

Alveolar integrity and ultrastructure in pigs remain undamaged after exposure to sevoflurane.

Previous studies have shown that both halothane and isoflurane have adverse but reversible effects on alveolar physiology. The present study was designed to test the hypothesis that also sevoflurane may affect alveolar integrity. Fifteen pigs were randomly selected to receive either thiopentone infusion (control group, n=8) or sevoflurane (n=7) at 4.0% inspiratory concentration (1.5 MAC) in air for 6 h. Tissue samples from the lungs were obtained at the end of the experiment. Both histopathological light microscopy and electron microscopy were used to assess the structural integrity of the alveoli. Pulmonary hemodynamics were comparable in both groups. Light microscopy showed no difference between the groups in the amount of alveolar macrophages, red blood cells or edema. Electron microscopy showed minor changes such as moderate local swelling of alveolar epithelium in both study groups. Alveolar type II cells were ultrastructurally unaltered in both study groups. We conclude that long-term, high concentration exposure to sevoflurane has no detrimental effect on the alveolar integrity in pigs.

Nucleolar morphometry in fine needle aspiration biopsies of the prostate.

OBJECTIVE: To find efficient morphometric nucleolar features for distinguishing different prostatic atypia groups in fine needle aspiration biopsies. STUDY DESIGN: A computerized interactive morphometry program was used to outline nucleoli of prostate cells. The samples were divided into three groups: group 1 (definitely benign [n = 10] and atypical but benign [n = 13]), group 2 (moderately atypical [n = 11] or highly suspicious [n = 13]) and group 3 (definitely malignant [n = 17]). RESULTS: The analysis revealed a difference in the number of nucleoli between definitely benign samples and other atypia groups but not between the latter. Nucleolar size features were best in distinguishing between atypia groups. The sample-associated mean size features were more powerful than cell group-associated size features. CONCLUSION: The sample-associated mean area, defined from cells selected by an experienced cytologist, if larger than 2 micron 2, was strongly associated with definitely malignant samples. The same was true for the largest nucleolar area if larger than 5.0 micron 2. Morphometric nucleolar size features appeared efficient in distinguishing between definitely malignant and other samples.

Expression patterns of cathepsins B, H, K, L and S in the human endometrium.

Cathepsins B, H, K, L and S belong to the family of lysosomal cysteine proteinases and participate in a variety of proteolytic processes, including degradation of the extracellular matrix (ECM). In the present study, we used Northern hybridization to demonstrate the presence of mRNAs for cathepsins B, H, K, L and S in human endometrium during both the proliferative and secretory phases of menstrual cycle. The mRNA levels for cathepsins H and K were significantly lower in secretory phase endometrium in comparison with proliferative phase endometrium. Immunohistochemical localization of the different cathepsins revealed widespread distribution of all cathepsins in both stroma and epithelial cells. The immunoreactivity for cathepsins B, H and K exhibited changes related to endometrial location and/or to the phase of the cycle. The strongest immunoreactivity for cathepsins B, H, L and S was observed in the surface epithelium of the endometrium. The staining for cathepsins was predominantly intracellular, but immunoreactivity was also detected on the surface of small lymphoid cells in the stroma. The findings of the present study suggest that cysteine cathepsins are needed for normal development and function of human endometrium during both the proliferative and secretory phases.

Placental expression of syndecan 1 is diminished in preeclampsia.

OBJECTIVE: Syndecan 1 is a cell surface heparan sulfate proteoglycan that binds growth factors and antithrombin III. The objective of this study was to examine whether placental expression of syndecan 1 in preeclampsia differs from that in normal pregnancy and whether gestational age and fetal growth affect syndecan 1 expression. STUDY DESIGN: An immunohistochemical analysis of 30 placentas of women with severe preeclampsia and 15 placentas of women without preeclampsia was performed with the monoclonal anti-syndecan 1 antibody B-B4. RESULTS: In 47% of preeclamptic placentas the immunoreactivity with antibody B-B4 was faint or absent, whereas 93% of the normal placentas exhibited strong immunoreactivity. The reduction in placental expression of syndecan 1 in preeclampsia was not associated with gestational age or impaired fetal growth. CONCLUSION: The expression of syndecan 1 on the chorionic villi is reduced in preeclampsia irrespective of gestational age or fetal growth.

Detection of human papillomavirus (HPV) in laryngeal carcinoma cell lines provides evidence for a heterogeneic cell population.

The role of human papillomavirus (HPV) has been studied in laryngeal carcinomas with contradictory results. To evaluate the causal relationship between HPV infection and epithelial malignancies of the larynx, 27 laryngeal carcinoma cell lines from 22 patients were studied. Also, paraffin-embedded biopsy samples of the original tumours were available from 12 patients. First, Southern blot hybridisation (SBH) was used for the analysis of 18 cell lines and 12 original tumour sections were studied by in situ hybridisation (ISH) to detect HPV. Further, cell lines and tumour biopsy samples were investigated with polymerase chain reaction (PCR) using three sets of consensus primers directed to L1 and E1 ORFs (open reading frames) and type-specific primers to HPV 16 E6 region. The adjacent apparently normal epithelium of one original biopsy sample showed positive signals for HPV by ISH. All other samples were HPV negative with these methods. The study was then extended to 27 laryngeal carcinoma cell lines, including the 18 cell lines studied earlier. A new nested PCR method was used with MY as external and general primers (GP) as internal primers for the cell lines and original tumour samples to achieve a maximal sensitivity. Subsequent SBH was performed to confirm the specificity of PCR products with both low- and high-risk HPV oligonucleotide probe mixtures and also with the HPV 16 oligoprobe. With this method, seven of 27 (26%) cell lines and seven of 12 (58%) tumour samples were found to harbour high-risk HPV. In two cases both the original tumour sample and the derived cell line showed HPV positivity. These results indicate that HPV copy numbers are low and only a minority of tumour cells harbour HPV DNA, explaining partly the controversial results reported earlier.

Expression of nitric oxide synthase in normal and preeclamptic placental tissue and effects of glyceryl trinitrate and shear stress on placental blood flow.

BACKGROUND: To study the expression of constitutively expressed nitric oxide synthase (cNOS) as well as the effects of glyceryl trinitrate (GTN) and shear stress on normal and preeclamptic placental tissue. METHODS: The expression of cNOS was studied using NADPH diaphorase activity reaction in seven normal and four preeclamptic placentas. The effect of GTN (n = 5) and shear stress induced by increasing the flow rate in the perfusion system (n = 5) was studied using an in vitro placental perfusion method. RESULTS: No difference in the distribution of cNOS in placental tissue was found between preeclamptic and normal pregnancies. Shear stress did not affect the production of nitric oxide metabolites. GTN was able to dilate placental vasculature. CONCLUSIONS: cNOS derived from syncytiotrophoblasts may not contribute to the development of preeclampsia. Placental vasculature responds to nitric oxide by vasodilation.

Expression of syndecan-1 in human placenta and decidua.

Syndecan-1 is a cell surface heparan sulphate proteoglycan, which binds to the extracellular matrix (ECM), growth factors and antithrombin III. The early expression of syndecan-1 during mouse embryonic development suggests a potential role in the communication between the embryo and the ECM of decidua. Using immunohistochemical methods, the present study showed that the expression of syndecan-1 in the trophoblast cells changes along trophoblast differentiation. The syncytiotrophoblasts in the chorionic villi exhibited an apical expression of syndecan-1. This suggests that the expression is restricted to non-migrating, non-proliferating trophoblasts. The mode of syndecan-1 expression by human placental trophoblasts is independent of gestational age. The expression is not changed in miscarriages. In pre-eclampsia, the staining for syndecan-1 on the villous syncytiotrophoblast is weaker compared to normal pregnancy, but in placental bed the expression is similar. The unique apical localization of syndecan-1 in chorionic villi, not detected in any other tissues, suggests a potential role in fetomaternal communication probably via growth factor binding and in anticoagulation of intervillous circulation.

Characteristics of Helicobacter pylori-negative and -positive peptic ulcer disease.

OBJECTIVE: To examine histological and other features of patients with Helicobacter pylori-positive (HP+) and -negative (HP-) peptic ulcers and characterize typical features of peptic ulcer in elderly patients. METHODS: 137 consecutive patients with peptic ulcer and 70 patients with dyspepsia were studied over 24 months. Endoscopy and a structured personal interview were carried out in all patients. Three histological specimens were obtained from the antral mucosa. RESULTS: The mean age of ulcer patients was 62.4 (SD 15.7) years and that of dyspepsia patients was 56.9 (SD 18.1) years (P=0.026). Of patients with HP- ulcers, 80.6% were aged 65 years or over; the corresponding proportion of patients with HP+ ulcer was 33.7% (P=0.0001). The use of non-steroidal anti-inflammatory analgesics was more common among those whose ulcer was HP- (69.4% vs 33%, P=0.0002). The occurrence of antral inflammation, atrophy and intestinal metaplasia did not differ between those with HP- and HP+ ulcers, but activity of gastritis was more common in HP+ than in HP- patients (94.9% vs 47.1%, P=0.0001). In the logistic regression analysis, HP- peptic ulcer disease was independently associated with older age, bile reflux, the use of non-steroidal anti-inflammatory analgesics and intestinal metaplasia, while HP+ ulcer disease was associated with active inflammation of the antral mucosa and tendency to ulcer recurrence. CONCLUSION: HP+ and HP- peptic ulcers have well-defined characteristics which probably reflect their different pathogenesis. Peptic ulcer disease of elderly people is mostly HP-.

Readmitted nonpsychotic patients and their eventual diagnoses. A retrospective study on 64 first-timers of inpatient care in the Psychiatric Clinic of Turku, Finland.

This longitudinal study was performed to characterise frequently readmitted patients in a sample of 64 first-timers of inpatient care. Half of the 12 revolving door patients were psychotic at last discharge. The relative risk for diagnostic change in the Axis I group was nine times higher than in the personality disorder group.

Tumour necrosis factor-alpha selectivity modulates expression of collagen genes in rat granulation tissue.

OBJECTIVE: To study the effects of tumour necrosis factor-alpha (TNF-alpha) on granulation tissue in rats. DESIGN: Animal experiment. SETTING: University Hospital, Finland. MATERIAL: 48 rats in 6 groups. INTERVENTIONS: The test implants were treated with either a single or daily injections of 200 ng of TNF-alpha. The samples were collected 4 and/or 7 days postoperatively. MAIN OUTCOME MEASURES: Pro alpha 1 (I) and pro alpha 1 (III) collagen mRNA concentrations, hydroxyproline, nitrogen, uronic acids, and hexosamine content of the sponge; and ingrowth of new granulation tissue into the sponge. RESULTS: A single application of TNF-alpha resulted in no significant differences between the groups. After daily applications of TNF-alpha the accumulation of collagen hydroxyproline and volume of ingrowth of granulation tissue were significantly lower than in the controls. After 7 days there were significant reductions in the concentrations of RNA-ribose, nitrogen, and uronic acids. Daily injections of TNF-alpha reduced pro alpha 1 (I) collagen mRNA concentrations but those of pro alpha 1 (III) collagen mRNA levels were not lowered to the same extent. CONCLUSION: Repeated application of TNF-alpha was required to reduce the synthesis of collagen. This down-regulation was selective as pro alpha 1 (I) collagen mRNA concentrations were lowered more than those of pro alpha 1 (III) collagen mRNA.

Standardized mitotic counts in breast cancer. Evaluation of the method.

Twenty-one pathologists and technicians participated in a study evaluating the variation present in mitotic counts for prognostication of breast cancer. The participants counted the mitotic figures in 20 breast cancer samples from ten high power fields (mitotic activity index, MAI, giving the results in mitotic figures per 10 fields) and also made a correction for field size and area fraction of the neoplastic epithelium to get the standardized mitotic index (volume fraction corrected mitotic index, or M/VV index, giving the result in mitotic figures per square mm of neoplastic epithelium). The difference in variation between the two methods was not big, but the standardized mitotic index (SMI) showed consistently smaller variation among all participants and different subgroups. Experienced pathologists had the highest variation in mitotic counts, and specially trained technicians, the lowest. The efficiency of the mitotic counts in grading (the grading efficiency) was used to evaluate the mitotic counts. In groups without special training for mitotic counts the mean grading efficiency was lower (experienced and training pathologists both on average had the potential to grade 88% of the cases correctly) than in the group specially trained for the purpose (trained technicians had the potential to grade 95% of the cases correctly). Among the specially trained technicians, the grading efficiency was of the same magnitude as the grading efficiency achieved in determining the S-Phase fraction of cells from paraffin embedded breast cancers by flow cytometry in different laboratories. The results suggest that special training is helpful in making mitotic counts more reproducible, and that in trained hands, the mitotic counts give results comparable to more sophisticated methods of determining proliferative activity in breast cancer.

Section thickness and mitotic counts in ovarian mucinous carcinoma. Methodological study with scanning confocal microscopy.

To study the effect of section thickness on mitotic counts, paraffin sections of 28 cases of mucinous ovarian carcinomas were analyzed. Mitotic counts, estimated with the number of mitoses per mm2 of neoplastic epithelium (M/Vv index) and per mm3 from optical sections and through the whole section thickness were done. Section thickness was measured with scanning confocal microscopy from each specimen of 3 series of sections with different nominal thicknesses (5, 8, and 10 microns). Section thickness varied considerably within each series of sections. The sections of the 5-microns group tended to be thicker and the sections of the 10-microns group thinner than expected on the basis of their nominal values. As expected, mitotic counts through the section gave higher M/Vv index values than counts done using optical sections. M/Vv values obtained using optical sections increased with increasing nominal section thickness. The study suggests that, when the section thickness cannot be measured, it is advisable to do mitosis counting by using optical sections in lightly stained tissue sections in the thickness range of 8-15 microns.

Factors predicting readmissions in personality disorders and other nonpsychotic illnesses. A retrospective study on 64 first-ever admissions to the Psychiatric Clinic of Turku, Finland.

A retrospective study of factors predicting readmissions and follow-up treatment was undertaken of all first-ever episodes of inpatient care (age under 65), excluding psychotic and organic mental disorders, during 1987 and 1988 in University Psychiatric Clinic in Turku City Hospital, Finland. The cohort consisted of 64 subjects, 24 men and 40 women. The study was carried out in the end of 1993, thus allowing about 5 years of follow-up. The diagnosis of personality disorder did not predict readmission. The only factor predicting readmission nearly significantly was not having a relationship. The incidence of the revolving door syndrome, defined as 4 or more admissions within 5 years, was 12.5%. Women had a greater risk of readmission, but not that of the revolving door. Patients who had psychotherapy as follow-up treatment showed a frequency of 8% for 4 or more admissions, whereas patients who had no follow-up treatment had a frequency of 21% for 4 or more readmissions. The only factor significantly predicting follow-up treatment arrangement was previous treatment contact.