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Sixteen seminomas with surrounding tissue containing normal and precancerous (cis) seminiferous tubules were examined for the expression of Fas (CD95, APO‐1) and Fas ligand (FasL) (CD95L). This was done by analyzing frozen specimens using immunohistochemistry with antibodies directed against Fas and FasL. The study showed that varying numbers (mean approx. 20%) of Fas‐positive lymphocytes were present among tumor‐infiltrating lymphocytes, but very few FasL‐positive lymphocytes. Fas was not expressed by normal seminiferous tubules and only occasional Fas‐positive epithelial cells were seen in cis tubules. FasL was expressed in 9 out of 10 cases in virtually all normal seminiferous tubules, mainly as a thin layer at the base of the seminiferous epithelium. In precancerous tubules, this layer was discontinuous and less pronounced. Rete testis expressed FasL in 2 out of 2 cases with rete present and Fas in 1 out of 1 case. Invasive tumor cells did not express Fas or FasL. The data are discussed in relation to immune reactions to seminomas and to the concept of the testis being an immunologically privileged area.

Infarct kidney hypertension was induced in congenital athymic nude rats and in their haired normal littermates. In both groups a significant and similar elevation of blood pressure was observed. The mesenteric vessels were studied histologically five, 12 and 20 days after operation. Necrotizing vasculitis with and without perivascular inflammatory reactions was found in mesenteric arteries and arterioles in six out of six athymic and in six out of 11 control rats. In sham operated athymic rats and in haire'd littermates neither hypertension nor vasculitis was observed. These observations indicate that the thymus play no role in the pathogenesis of acute hypertensive vascular disease

The effect of chronic hypertension on cerebral blood flow (CBF) was studied in anaesthetised rats. CBF was measured with the intracarotid 133Xe injection method. Rats with spontaneous and renal hypertension were compared with normotensive controls. The lower limit of autoregulation was determined during controlled haemorrhage. In the normotensive rats, CBF remained constant until mean arterial pressure (MAP) had decreased to the range of 50–69 mm Hg. Thereafter, CBF decreased with each further decrease in MAP. In both types of hypertensive rats, CBF remained constant until MAP had decreased to the range of 70–89 mm Hg. Thus, a 20-mm Hg shift of the lower limit of CBF autoregulation was found in both spontaneous and renal hypertensive rats. A neuropathological study revealed ischaemic brains lesions in half of the hypertensive rats following hypotension, whereas only a single lesion was found in one of six normotensive rats. No ischaemic brain lesions were found in a control study in which CBF was shown to be stable over a 2 1/2-h period. In conclusion, hypertensive rats showed a shift of the lower limit of CBF autoregulation as well as an increased susceptibility to ischaemic brain damage during hypotension. These findings presumably reflect hypertensive structural changes in the cerebral circulation.

The human urinary bladder wall is traditionally described as being without a muscularis mucosae. A consecutive one year material of 772 bladder biopsies from 171 patients were examined for the proposed presence of lamina muscularis mucosae (MM). MM was observed in 15% of the biopsies and in 35% of the patients and graded into three patterns according to its continuity. Biopsies with transitional cell carcinomas were reviewed in order to find out whether MM-positive biopsies had been staged correctly. The data are discussed in relation to earlier studies on the subject.

12 patients with conservatively treated uremia were investigated using bone histomorphometry, bone aluminium concentration determination and total body bone mineral content (TBBM). The bone aluminium was raised in 10 patients and was significantly related to oral aluminium salt consumption (p<0.01). Two of four patients who had not received aluminium also had raised levels but the difference was not significant from nonuremic patients. The two patients with the highest levels had a mineralisation defect despite normal levels of 1, 25-dihydroxyvitamin D. Three patients had significant bone loss of whom one had osteomalacia (OM) while two had secondary hyperparathyroidism (2HP). It is concluded that 1) aluminium salt consumption results in bone aluminium accumulation, and may contribute to the mineralisation defect; 2) uremic patients not treated ith aluminium salts may have slightly raised levels, but this seems not to be clinically important; 3) secondary hyperparthyroidism causes greater destruction of bone mass than other uremic bone diseaes; 4) atomic absorption spectrometry is a more sensitive method for detecting aluminium bone deposition than histochemical methods.

We have studied the physical interaction between macrophages and lymphocytes during the immune response to purified protein derivative of tuberculin (PPD) in vitro. Mixtures of peritoneal macrophages and lymph node lymphocytes from guinea pigs immunized with tubercle bacilli formed cell clusters during 20 h of culture with PPD. The number of clusters produced was correlated to the number of immune lymphocytes in the cultures. Peritoneal macrophages which had been pulsed with PPD and untreated lymph node lymphocytes produced cell clusters in the absence of free PPD in numbers equivalent to those produced by the same cells in the presence of free PPD. In cultures containing a mixture of PPD-pulsed macrophages, not-pulsed macrophages, and immune lymphocytes with no free PPD, cell clusters developed mainly between the antigen-pulsed macrophages and lymphocytes. Cluster formation was antigen-specific with the specificity residing in the lymphocytes, mainly or exclusively in the T lymphocytes. These data indicate that in the process of cell cluster formation macrophages serve as antigen-binding (or -processing) cells, while a subpopulation of lymphocytes interact physically and specifically with the macrophages.

Macrophage-lymphocyte clusters are formed when lymph node cells and autologous peritoneal exudate cells from guinea pigs immunized with tubercle bacilli are cultured in the presence of purified protein derivative of tuberculin (PPD) for 20 h. We have studied the ultrastructure of these clusters employing transmission and scanning electron microscopy. The most simple macrophage-lymphocyte cluster consisted of one macrophage, one large central lymphocyte with a blastoid appearance attached to the macrophage with a broad area of contact, and from a few to more than 20 small peripheral lymphocytes attached to the central lymphocyte by their uropods. Some clusters were of more complex type, containing two or three macrophages or one macrophage with more than one central lymphocyte attached to the surface, but even in these clusters each peripheral lymphocyte was attached only to one central lymphocyte. By morphological criteria the peripheral lymphocytes were T lymphocytes.

Myelolipoma is a benign oligosymptomatic tumour of the adrenal gland. Clinical diagnosis and surgical removal are extremely rare. We report an operated case found incidentally by urography in a 52-year-old woman with a toxic hepatitis. The previously reported and operated cases are briefly reviewed.

Abstract: The differentiation of 39 diagnostic biopsies, as determined by histologic grading, and that of the corresponding 39 rectal carcinomas were compared. About 60 percent of grade I tumors were diagnosed by grade 1 biopsies, while about 90 percent of grade II and only about 40 percent of grade III tumors were diagnosed by grade II and grade III biopsies, respectively. The results indicate caution when biopsy grading is used as a guidance to therapy, especially of poorly differentiated tumors.

INTRODUCTION: In 10% of acute myeloid leukemia (AML) patients the recurrent balanced translocation t(8;21) places the RUNX1 (AML1) gene next to RUNX1T1 (ETO) gene resulting in the generation of a RUNX1-RUNX1T1 fusion protein. While in mouse models this protein has failed to produce leukemia alone, alternative splicing of exon 9a of the RUNX1-RUNX1T1 fusion transcript has recently (Yan et al, Nat Med, 12, 945, 2006) been shown to result in a truncated RUNX1-RUNX1T1 fusion protein with increased leukemogenic potential in a retroviral transduction-transplantation model. HYPOTHESIS: We hypothesized that i) the fusion of RUNX1 to RUNX1T1 alters exon 9a splicing balance, ii) Fusion transcripts containing exon 9a is involved in leukemogenesis in RUNX1-RUNX1T1 positive AML patients, and iii) the ratio between fusion transcripts with and without exon 9a may have prognostic significance. MATERIALS AND METHODS: Real-time quantitative PCR analysis was performed using primer-probe sets discriminating between the RUNX1-RUNX1T1 fusion transcript as well as the RUNX1T1 transcript containing exon 9a (R+9a) or lacking exon 9a (R-9a), respectively. Twenty-three diagnosis and 117 follow-up samples from 14 RUNX1-RUNX1T1 positive patients and diagnosis samples from 13 RUNX1-RUNX1T1 negative AML patients, as well as from 29 normal donors were included in the study. RESULTS: Levels of the native RUNX1T1 mRNAs were expectedly low in both healthy donors and RUNX1-RUNX1T1 negative AML patients, allowing us to define a threshold, above which R+9a and R-9a expression in RUNX1-RUNX1T1+ patients truly reflected RUNX1-RUNX1T1 fusion transcript expression. Thus, median RUNX1-RUNX1T1 expression was a factor 500 higher than the BM threshold and a factor 104 higher than the PB threshold. The ratios between R+9a and R-9a transcripts were within a relatively narrow range in normal donors and RUNX1-RUNX1T1 negative patients (0.017 to 0.11, median 0.06). By contrast, the R+9a/R-9a ratios in RUNX1-RUNX1T1 positive AML patients were significantly higher (range 0.05–0.46, median 0.17), (Wilcoxon’s rank sum test, P<0.001), with 12/14 patients displaying R+9a/R-9a ratios above the normal range in either bone marrow (BM) or peripheral blood (PB). More importantly, patients who achieved a continuous complete remission (CCR) displayed a more rapid disappearance of the RR+9a compared to RR-9a (Wilcoxon’s rank sum test of individual patient slopes BM: P=0.07, PB: P=0.014), further corroborating the notion that deregulated RUNX1-RUNX1T1 splicing is part of leukemogenesis (Figure). CONCLUSION: Deregulated RUNX1-RUNX1T1 splicing is part of the leukemogenic process in the majority of RUNX1-RUNX1T1 positive AML patients. We suggest that analysis of splice variant kinetics during cytoreduction may, if validated in larger studies, be a useful way to identify patients prone to relapse. Splice variant kinetics during cytoreductive treatment in BM (A) and PB (B). Black lines: patients with later relapse. Grey broken lines: patients remaining in CCR. X-axis: time after diagnosis, y-axis: RR+9a/RR-9a splice variant ratio relative to ratio at diagnosis. Figure Figure

BACKGROUND: Wilms’ Tumor gene 1 (WT1) is highly expressed in 80% of AML patients and has been proposed as a valuable tool for minimal residual disease (MRD) detection. Though pilot studies have shown the value of MRD monitoring using WT1 as marker, particularly during therapy, no single center study has addressed the value of close monitoring both during therapy and post-therapy follow-up or developed models to reliably use this information in clinical decision-making. AIMS: To determine whether high residual WT1 expression at the time where complete remission is first diagnosed (CR1) is an adverse prognostic factor, to evaluate the value of WT1 expression levels in peripheral blood (PB) and bone marrow (BM) for prediction of disease relapse, and to develop mathematical models that will enable quantification of the central parameters related to prediction of clinical relapse. METHODS: WT1 levels were quantified using real-time quantitative RT-PCR by normalization to the control genes B2M and ABL, and in follow-up samples expressed as a fraction of the BM diagnostic level. In a cohort of 165 adults and 18 children, treated at the Departments of Hematology and Pediatrics, Aarhus University Hospital, BM and PB samples from 90 patients (76 adults and 14 children) were analyzed at diagnosis and during therapy and follow-up (952 samples, median 8 samples/patient, range 2-38). Prognostic difference between groups was determined using the Cox Proportional Hazards statistical model including age, sex, cytogenetics, de novo/secondary leukemia, FLT3-ITD, and white blood cell counts. RESULTS: WT1 levels in BM and PB at CR1 above that of the average plus two standard deviations of 39 BM or 30 PB of healthy donors were found to be an independent adverse prognostic factor regarding both disease-free survival (BM: Hazard ratio (HR) 4.91, P=0.001, PB: HR 4.97, P=0.008) and overall survival (BM: HR 2.65, P=0.047, PB: HR 3.55, P=0.025). We were able to describe relapse kinetics in 29/37 relapses observed in the 90 patient cohort. By grouping 34 BM and 99 PB samples in monthly intervals prior to clinical relapse, disease development was delineated (Figure). Based on mathematical considerations involving proportions of the integrals of the graphs shown in the figure we were able to predict how sampling intervals influence relapse detection rates (RDRs) as well as the median time from WT1 positivity to clinical relapse (t(m)). For example, a BM sampling interval of 4 months will lead to a RDR of 93% and a t(m) of 74 days. A PB sampling interval of 2 months will lead to a RDR of 81% and a t(m) of 44 days. CONCLUSIONS: CR1 WT1 expression levels in both BM and PB are independent prognostic factors in AML. Though relapse was seen earlier in BM than in PB our models demonstrates that frequent PB sampling provides an acceptable window of opportunity for initiation of preemptive cytoreduction as a consequence of molecular relapse. Relapse prediction in monthly intervals; x-axis: time, relapse at t=0, y-axis: samples with WT1 level above threshold as a fraction of all samples. Median number of relapses in each interval; BM: 7(range 6–9), PB: 9(range 6–13) Relapse prediction in monthly intervals; x-axis: time, relapse at t=0, y-axis: samples with WT1 level above threshold as a fraction of all samples. Median number of relapses in each interval; BM: 7(range 6–9), PB: 9(range 6–13)

Aims: Myelodysplastic syndromes (MDS) constitute a heterogeneous group of disorders with hematopoiesis in disarray and with preponderance to transformation to AML. In contrast to the situation in de novo AML patients, the hematopoiesis in MDS patients is often though to encompass most cell lineages with few non-malignant cells in the bone marrow due to displacement by the MDS clone. While the involvement of the myeloid sublineages has been relatively easy to verify in semi-solid CFU assays, that of the lymphoid lineages has required advanced flow sorting techniques in combination with FISH assays in the minority of MDS patients with a well-known genetic aberration such as 5q-, -7 or +8. Moreover, the latter studies have usually been restricted to the description of the involvement of the B-cell lineage. Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including MDS. In a patient with a very immature MDS that was found to be hypermethylated in the p15, HIC, E-cadherin, and Estrogen receptor genes, we wanted to elucidate the methylation status in both myeloid and lymphoid cells. Material and methods: Mononuclear cells from a 69-year-old female MDS patient with RAEB was analyzed. Using magnetic cell sorting, CD34 and CD3 positive cells were isolated from BM-MNC and PB-MNC, respectively. The purity of the isolated cell fractions was determined by flow cytometry (CD34+: 95% and CD3+: 84%). DNA isolated from the cells was treated with sodium bisulfite and analyzed for promoter hypermethylation in the p15, HIC, E-cadherin, and Estrogen receptor genes by means of Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE). Results: All cell fractions were found to be hypermethylated in the four genes analyzed. Since the Bisulfite-DGGE allows for the detection of heterogeneous methylation patterns, we were able to compare these patterns. Similar Bisulfite-DGGE band patterns were observed in total BM-MNC, total PB-MNC, CD34+ cells and CD3+ cells. Conclusion: Because the methylation band patterns were the same in all cell fractions, it seems unlikely that methylation has occurred independently in progenitors committed for myeloid and lymphoid maturation, respectively. We therefore conclude that promoter hypermethylation in this patient has occurred in a stem cell with potential for both myeloid and lymphoid maturation. This methodology should provide an easy way to assess the extent of clonality of hematopoiesis in MDS patients.