Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children.

AIMS/HYPOTHESIS: Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1-11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes. METHODS: The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1-3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence. RESULTS: Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1ß, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls. CONCLUSIONS/INTERPRETATION: These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.

Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors.

TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.

Risk of bilateral testicular germ cell cancer in Denmark: 1960-1984.

The incidence of a second primary testicular germ cell cancer among 2850 (96.6% of eligible) men with a histologically verified first primary germ cell cancer diagnosed in the period 1960-1979 in Denmark was established. Of these 2850 men, 73 (2.6%) developed a contralateral testicular cancer. In five of these patients (0.18%), the tumors were synchronous. The cumulative risk of developing a contralateral cancer 25 years after diagnosis of the first testicular germ cell cancer was 5.2% according to a Kaplan-Meier estimate. It was higher among men with a nonseminoma as the first tumor (8.4%) than among men with a seminoma as the first tumor (3.6%). Of the second tumors, 12% were stage II and 17% were stage III at the time of diagnosis. Based on 24,588 person-years at risk and 68 nonsimultaneously occurring bilateral testicular germ cell cancers, the overall relative risk (RR) of developing a second primary cancer in the contralateral testicle following a first germ cell cancer was found to be 24.8 (95% confidence interval = 19-38). Among men with a nonseminoma, the risk was higher (RR = 27.1) than among men with a seminoma (RR = 22.5). The excess risk was not affected by age at diagnosis, calendar period, or time since diagnosis. Close surveillance by screening for and treatment of carcinoma in situ of the remaining testicle in testicular cancer patients are advised.

Long-term fertility and Leydig cell function in patients treated for germ cell cancer with cisplatin, vinblastine, and bleomycin versus surveillance.

Fertility and Leydig cell function were investigated in 31 patients previously treated for nonseminomatous testicular cancer. Twenty-two patients with metastatic cancer had received cisplatin-based chemotherapy, and the median follow-up was 64 months (range, 42 to 100 months). Nine patients without metastases were treated with orchiectomy alone, and follow-up in this group was a median of 61 months (range, 40 to 77 months). None of the patients have relapsed and retroperitoneal lymph node dissection was not performed in any patient. Both the concentration of spermatozoa and the volume of the remaining testis are significantly reduced in patients who had previously received chemotherapy when compared with patients treated with orchiectomy alone (P less than .05). There were no significant differences between groups when comparing morphology, motility, and penetration of the spermatozoa. Subclinical Leydig cell dysfunction with normal testosterone and elevated luteinizing hormone (LH) was observed in one patient (11%) treated with orchiectomy alone, while 59% of the patients who had received chemotherapy had elevated LH (P less than .05). We conclude that cisplatin-based chemotherapy leads to a persistent impairment of fertility and Leydig cell function in the majority of patients with testicular cancer.

Impaired testicular function in patients with carcinoma-in-situ of the testis.

PURPOSE: To elucidate the biologic association between germ cell neoplasia and testicular dysfunction, through investigation of Leydig cell function and semen quality in men with carcinoma-in-situ (CIS) of the testis. PATIENTS AND METHODS: We examined two groups of men, unilaterally orchidectomized for testicular cancer. Biopsy of the contralateral testis had showed CIS in a group of 24 patients and no evidence of CIS in the other group of 30 patients. Semen quality and serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared in these two groups of men after orchidectomy but before further treatment for testicular cancer. RESULTS: Significantly higher LH levels (median, 8.1 IU/L v 4.8 IU/L; P < .001) and generally lower testosterone levels (median, 12.5 nmol/L v 15.5 nmol/L; P = .13) were found in the CIS group. The proportion of patients with Leydig cell dysfunction was higher in the group of patients with CIS (11 of 24) than in the group of patients without (two of 30) (P = .01). Sperm concentration and total sperm count were significantly lower (P < .001) in patients with CIS (median, 0.03 x 10(6)/mL and 0.10 x 10(6), respectively) than in patients without (median, 9.1 x 10(6)/mL and 32 x 10(6), respectively), whereas the levels of FSH were significantly higher (P < .001) in the former group of men (median, 19.6 IU/L v 9.0 IU/L). CONCLUSION: Not only spermatogenesis but also Leydig cell function is impaired in testes with CIS. This impairment could be due to common factors in the pathogenesis of germ cell neoplasm and testicular dysfunction. Alternatively, CIS cells may have a negative impact on Leydig cell function.

The PBX-regulating protein PREP1 is present in different PBX-complexed forms in mouse.

Human PREP1, a novel homeodomain protein of the TALE super-family, forms a stable DNA-binding complex with PBX proteins in solution, a ternary complex with PBX and HOXB1 on DNA, and is able to act as a co-activator in the transcription of PBX-HOXB1 activated promoters (Berthelsen, J., Zappavigna, V., Ferretti, E., Mavilio, F., Blasi, F. , 1998b. The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperatity. EMBO J. 17, 1434-1445; Berthelsen, J., Zappavigna, V., Mavilio, F., Blasi, F., 1998c. Prep1, a novel functional partner of Pbx proteins. EMBO J. 17, 1423-1433). Here we demonstrate the presence of DNA-binding PREP1-PBX complexes also in murine cells. In vivo, PREP1 is a predominant partner of PBX proteins in various murine tissues. However, the choice of PBX family member associated with PREP1 is largely tissue-type specific. We report the cloning and expression domain of murine Prep1 gene. Murine PREP1 shares 100% identity with human PREP1 in the homeodomain and 95% similarity throughout the whole protein. In the adult mouse, PREP1 is expressed ubiquitously, with peaks in testis and thymus. We further demonstrate the presence of murine Prep1 mRNA and protein, and of different DNA-binding PREP1-PBX complexes, in mouse embryos from at least 9.5 days p.c. Moreover, we show that PREP1 is present in all embryonic tissues from at least 7.5-17.5 days p.c with a predominantly nuclear staining. PREP1 is able to super-activate the PBX-HOXB-1 autoregulated Hoxb-1 promoter, and we show that all three proteins, PREP1, PBX and HOXB-1, are present together in the mouse rhombomere 4 domain in vivo, compatible with a role of PREP1 as a regulator of PBX and HOXB-1 proteins activity during development.

Direct interaction of two homeoproteins, homothorax and extradenticle, is essential for EXD nuclear localization and function.

The Drosophila Homothorax (HTH) and Extradenticle (EXD) are two homeoproteins required in a number of developmental processes. EXD can function as a cofactor to Hox proteins. Its nuclear localization is dependent on HTH. In this study we present evidence of in vivo physical interaction between HTH and EXD, mediated primarily through an evolutionarily conserved MH domain in HTH. This interaction is essential for the mutual stabilization of both proteins, for EXD nuclear localization, and for the cooperative DNA binding of the EXD-HTH heterodimer. Some in vivo functions require both EXD and HTH in the nucleus, suggesting that the EXD-HTH complex may function as a transcriptional regulator.

Localized irradiation of testes with carcinoma in situ: effects on Leydig cell function and eradication of malignant germ cells in 20 patients.

Twenty men (median age, 31 yr) previously treated for unilateral testicular cancer received localized irradiation in a dose of 20 Gray in 10 fractions for carcinoma in situ of the remaining testis. Follow-up testicular biopsies performed 3 (n = 19) and 24 (n = 14) months after the treatment showed in all cases a Sertoli cell-only pattern. Hormonal evaluation was performed before as well as 3, 12, 24, and 36 months after radiation treatment. Endocrine parameters were followed for a median of 30 months (3-36 months). Baseline serum testosterone values decreased during the follow-up period from 13.3 +/- 6.0 to 10.8 +/- 6.4 nmol/L (mean +/- SD), although the decrease was not statistically significant (P = 0.06). Serum LH values increased during the first 3 months of follow-up from 10.4 +/- 5.4 to 15.6 +/- 7.3 IU/L (P less than 0.0001) and then remained unchanged. Significant decreases in GnRH- and hCG-stimulated testosterone levels also indicated an impairment of Leydig cell function. FSH levels increased (P less than 0.0001) during the first 3 months of follow-up from 21.8 +/- 11.1 to 33.2 +/- 13.2 IU/L. We conclude that localized irradiation of 20 Gray eradicated carcinoma in situ germ cells. Development of a second testicular cancer has until now been prevented. Leydig cell function was partially impaired by the radiation dose given.

Cloning and expression of human NF-YC.

The CCAAT box is an important element in eukaryotic promoters and NF-Y (CBF) is a conserved heterotrimeric protein binding to it. Two subunits, NF-YB and NF-YC, contain a histone-like motif. We cloned the complete cDNA coding for the human NF-YC gene. The ORF codes for a 335 aa protein that shows virtual identity to the rat sequence, confirming the stunning invariance of NF-Y genes across species. We expressed and purified the yeast homology domain of NF-YC in bacteria and performed EMSA together with the corresponding conserved domains of NF-YA and NF-YB, obtaining a CCAAT-binding mini-NF-Y. We evaluated the expression of NF-YC and found that mRNA levels are similar in different human tissues except in testis.

Ultrastructure of meiotic chromosomes in boys undergoing chemotherapy for leukemia.

Meiotic chromosomes from four boys treated with combination chemotherapy for acute lymphoblastic leukemia were analysed ultrastructurally by serial sectioning and reconstruction of spermatocyte chromosomes. Analysis of the 1012 chromosomes in 22 zygotene and pachytene spermatocyte nuclei showed that in most of the cells that reached the pachytene stage, one or more of the following abnormalities were found in the intranuclear structures: decreased condensation of euchromatin, centromeric heterochromatin, diffuse heterochromatin, or abnormal nucleolar morphology. The total number of recombination nodules and -bars was within normal limits, but in some nuclei the ratio between the two types of structure was inappropriate for the stage, indicating developmental asynchrony. With the exception of one nucleus, there was no increase in abnormalities of pairing and synaptonemal complex formation. The number of structural aberrations in autosomal and sex chromosomes was similar to that found in the reference material. The repair system responsible for resolving chromosome interlockings in zygotene was functioning. It is not possible to predict which of the short term effects found will become permanent and thus pose a risk of genetically abnormal conceptions. New means of classifying meiotic chromosomes, of evaluating the synchrony of development, and of analysing the telomere attachment pattern are described.

Sperm counts and serum follicle-stimulating hormone levels before and after radiotherapy and chemotherapy in men with testicular germ cell cancer.

Sperm counts were low (median, 15 X 10(6) per ejaculate) and serum follicle-stimulating hormone (FSH) levels were moderately elevated (median, 31 IU/l) after unilateral orchiectomy and immediately before radiotherapy and chemotherapy in 34 patients with seminomas and 20 patients with nonseminomatous germ cell tumors. The scattered radiation (0.2 to 1.3 Gray [Gy]) reaching the remaining testicle during radiotherapy caused azoospermia in more than two thirds of the patients. A median of 540 days elapsed after the end of treatment before spermatozoa were again found in semen samples, while a median of 1250 days passed before the pretreatment sperm count was reached. One to 5 years after treatment, sperm counts were still low (median, 6 X 10(6) per ejaculate) and serum FSH was elevated (median, 61 IU/l). The adjuvant chemotherapy given to the 20 patients with nonseminomatous tumors did not appear to affect restitution appreciably.

Gonadal function in men with testis cancer.

A group of 218 patients with unilateral germinal testicular cancer was investigated with biopsy from the contralateral testis and/or semen and hormone analyses after orchidectomy but before irradiation and chemotherapy. In 24% of the biopsies severe irreversible changes such as spermatogenic arrest, Sertoli cell-only tubules, hyalinized tubules, or carcinoma in situ were found. Serum testosterone (T) values were below the reference interval in 13% of the patients, whereas 12% had normal serum T values in combination with elevated serum luteinizing hormone (LH). Most serum follicle-stimulating hormone (FSH) values were above the reference interval. The impairment of gonadal function in the patients could be explained partly by the removal of one of the testes, by premorbid defects of spermatogenesis in the contralateral testis, and by elevated serum human chorionic gonadotropin (hCG) values caused by the cancer.

Acute effects of graded alcohol intake on glucose, insulin and free fatty acid levels in non-insulin-dependent diabetic subjects.

Alcohol-induced hypoglycaemia is a well-known phenomenon in insulin-treated diabetic subjects. Less attention has been paid to the impact of alcohol on blood glucose and insulin responses in non-insulin dependent diabetic subjects. The aim of this study was to investigate the acute metabolic effects of different alcohol contents added to a non-alcohol beer in 10 non-insulin-dependent diabetes mellitus (NIDDM) subjects. The patients received 500 ml non-alcohol beer with an alcohol percentage (v/v) of 0 (A), 2.7 (B), and 5.4 (C), implying identical contents of ingredients except for alcohol. Blood glucose (mean +/- SE) responses were similar in the three situations (395 +/- 59, 365 +/- 86 and 261 +/- 26 mmol/l x 240 min). In contrast, the incremental insulin response areas increased dose dependently to alcohol (5430 +/- 1158, 9336 +/- 2172 and 12336 +/- 2922 pmol/l x 240 min) and showed a linear correlation (r = 0.39; P < 0.03). The average suppression of serum free fatty acid was similar in the three situations (72.4 +/- 4.4%, 76.3 +/- 6.0% and 68.2 +/- 6.3%). In conclusion, intake of small amounts of alcohol does not acutely deteriorate the glycaemic control in NIDDM. The fact that alcohol results in a dose-related elevation in insulin levels with unaltered blood glucose and free fatty acid responses in NIDDM points to an aggravation of insulin resistance.

Intratubular germ cell neoplasia (carcinoma in situ): a preinvasive lesion of the testis.

The existence of a preinvasive lesion of the testis, referred to as intratubular germ cell neoplasia or carcinoma in situ, is now well established. The finding has been made in infertile males with various types of testicular abnormalities. In a number of cases, there has been subsequent progression to typical malignant germ cell tumors, including seminoma and embryonal carcinoma. Light and electron microscopic studies indicate that the abnormal germ cells within the seminiferous tubules resemble germ cells at early stages of differentiation. The cells lack intercellular connections characteristic of normal developing germ cells, but otherwise are similar to gonocytes and prespermatogonia in the fetal and prepubertal testis.

Internal parasite levels and response to anthelmintic treatment by beef cows and calves.

Albendazole (methyl 5-propylthio-1 H-benzimidazol-2-yl carbamate) was used as an anthelmintic in a 3-yr study involving 578 beef cows and 438 nursing calves. Infection levels for nematodes, coccidia, and tapeworm were relatively low throughout the 3-yr period. Eggs per gram of feces in cows and calves were lower (P less than .01) 2 wk posttreatment but were not different 5 mo later, when calves were weaned. Cow weight gain, rate and time of conception, and adjusted calf weaning weights were not affected significantly by deworming of either cows or calves. Level of nematode infection measured as eggs per gram of feces was higher (P less than .01) in younger cows than in mature cows. Although deworming with Albendazole lowered (P less than .01) nematode infection levels, no responses were observed in cow or calf performance.

Prevalence of carcinoma in situ of the testis in 207 oligozoospermic men from infertile couples: prospective study of testicular biopsies.

OBJECTIVE: To investigate the prevalence of carcinoma in situ of the testis in a group of oligozoospermic men from infertile couples. DESIGN: A consecutive group of oligozoospermic men from infertile couples were offered bilateral testicular biopsy. The observed prevalence of carcinoma in situ was compared with the expected prevalence of testicular cancer in a corresponding age matched population of Danish men, assuming all untreated cases of carcinoma in situ progress to tumour stage. This calculation was based on data from the Danish Cancer Registry. SUBJECTS: 207 men aged 18-50 years who had sperm density below 10 million/ml in two samples within the previous 2 years or sperm density below 20 million/ml in two samples within the previous 2 years and a history of cryptorchidism or one or two atrophic testicles (orchidometer volume less than 15 cm3), or both. INTERVENTIONS: Bilateral testicular biopsies. MAIN OUTCOME MEASURES: Carcinoma in situ in the biopsy specimen. RESULTS: No case of carcinoma in situ was found among the 207 men. The expected number in a normal age matched population of corresponding size was 0.8. CONCLUSIONS: There is no increase in risk of carcinoma in situ of the testis in moderately oligozoospermic men of couples referred because of infertility.

[Irrigation of vas deferens during vasectomy]. / Gennemskylining af vas deferens under vasektomi

PIP: 59 men underwent vasectomy under local anesthesia. In 29 cases the ligated vas deferens was irrigated with 40 ml sterile water. Samples from every 4th ejaculate were examined, both with and without centrifuging at 2000 revolutions per minute for 5 minutes, to determine the number of spermatozoa. Centrifuging the samples gave significantly more reliable results (2P .005). Irrigation of the ligated vas deferens did not hasten the onset of azoospermia. No spontaneous recanalizations occurred.^ieng

[Ogilvie syndrome after Cesarean section]. / Ogilvies syndrom efter sectio.

INTRODUCTION: Ogilvie's syndrome, acute pseudo-obstruction of the colon, can lead to perforation of the caecum and death. The syndrome is not well known and diagnosis can be difficult to make in time. METHODS: We analysed seven cases of Ogilvie's syndrome with the aim of improving diagnostics. RESULTS: All had prolonged labour before cesarean section, which was complicated by bleeding. All were treated with syntocinon, a hormone that may influence gastrointestinal motility. All patients developed abdominal meteorism within a few days of operation, which increased despite the passing of flatus and stool. Five cases resulted in caecum perforation before the correct diagnosis and treatment were made. Perforation occurred on days 3-4, day 5, or probably days 8-10 after the operation. One of these patients, who suffered from severe adipositas, died. CONCLUSION: It is very important to make an early diagnosis, as the condition can progress quickly. Diagnosis should be made on the history, clinical assessment, and abdominal X-ray. Intermittent flatus and stool are characteristic of this truly non-obstructive condition and should not therefore delay a diagnostic X-ray.