Cognitive performance and attachment patterns at four years of age in extremely low birth weight infants after early intervention.

This study aims at assessing the effects of an early occupational therapy intervention on the cognitive development and the development of attachment patterns in ELBW infants. The intervention, given weekly at home from six months to 12 months, aimed at supporting parent-child interaction and enhancing motor control and coordination. The study population consisted of 100 ELBW infants matched in pairs in accordance to their pre-perinatal risk scores and allocated successively to intervention or non-intervention groups. Cognitive development was assessed with the Bayley Scales at age two and with the WPPSI at age four. Attachment to primary caregiver was assessed with the Preschool Assessment of Attachment (PAA). Cognitive performance was within age norms in both groups at both ages. Intervention did not show any effect on cognitive performance at the age of two years. At the age of four years, cognitive level was overall, and most notably for verbal performance, higher in the intervention group than in the control group. There was an over-representation of the so-called atypical attachment patterns (those not fitting the normative A, B, or C categories) in the control group. The results are discussed in terms of finding more global ways to support the development of at risk pre-term children.

Promoter function of different lengths of the murine luteinizing hormone receptor gene 5'-flanking region in transfected gonadal cells and in transgenic mice.

The purpose of these studies was to explore the sex- and tissue-specific expression of the LH receptor (LHR) gene. Fusion genes containing three different lengths of the 5'-flanking region of the mouse LHR gene (7.4 kb, 2.1 kb, and 173 bp), beta-globin intron, and the beta-galactosidase (beta-GAL) reporter gene were constructed. Function of these fusion genes [LHR (7.4 kb)/beta-GAL, LHR (2.1 kb)/beta-GAL, and LHR (173 bp)/beta-GAL] was studied in vitro and in vivo. beta-GAL expression was higher in transfected mouse Leydig (mLTC-1) than in granulosa (KK-1) tumor cells with all three constructs. The shortest LHR (173 bp)/beta-GAL construct showed the highest level of beta-GAL expression in both cell types. beta-GAL expression was clearly suppressed with the 2.1-kb promoter and was nearly undetectable with the 7.4-kb construct. In transgenic mice, all three constructs directed beta-GAL expression to adult Leydig cells, displaying decreasing intensity with increasing promoter length. Unexpectedly, beta-GAL expression was also found in elongating spermatids, but not in fetal Leydig cells. There was no expression in any ovarian cell type with the three constructs used, except that one of five mouse lines with the LHR (7.4 kb)/beta-GAL construct expressed beta-GAL in their thecal cells. Two lines transgenic for the 7.4- and 2.1-kb promoter constructs each directed high beta-GAL expression to the brain, with higher intensity in 7.4-kb lines. All promoters directed expression to the pituitary gland, some faintly to the adrenal gland. Northern hybridization analysis of the beta-GAL transcripts in Leydig cells revealed that the 173-bp promoter mainly gave rise to the full-length beta-GAL messenger RNA, whereas the 2.1- and 7.4-kb promoters mainly induced transcription of truncated beta-GAL messages. This suggests that the 5'-flanking region, upstream of -173, determines the formation of splice variants of the structural gene to be transcribed. The present findings in transgenic mice provide in vivo evidence for basal transcriptional activity of the first 173 bp upstream of the LHR translation initiation codon. In conclusion, the promoter function of the mouse LHR 5'-flanking region is tissue, age, and sex specific. The sequence upstream of the basal promoter determines extragonadal LHR expression as well as the alternate splicing of its message. The promoter sequences directing LHR expression to fetal Leydig cells and ovary reside outside the 7.4-kb 5'-flanking region and remain to be identified.

Age- and sex-specific promoter function of a 2-kilobase 5'-flanking sequence of the murine luteinizing hormone receptor gene in transgenic mice.

A transgenic (TG) mouse model carrying a 2-kb murine LH receptor (LHR) promoter/beta-galactosidase (beta-GAL) fusion gene was created to study the regulatory function of the 5'-flanking region of the murine LHR gene. Of the five TG mouse lines produced, three displayed high beta-GAL expression in the testis, but none showed any expression in the ovary. In addition, all mouse lines of both sexes expressed beta-GAL consistently in the brain, most prominently in hippocampus, hypothalamus, midbrain, and cortex. Weak staining was found in a few pituitary samples. All other tissues examined were negative for transgene expression. In support of sex-specific gonadal expression of the transgene, transient transfection of the LHR/beta-GAL gene construct into immortalized mouse granulosa (KK-1) and Leydig (mLTC-1) tumor cells revealed a more than 5-fold higher expression level in the Leydig cells. Histological examination of the TG testes demonstrated strong beta-GAL expression in Leydig cells, but, unexpectedly, also in elongating spermatids of adult age and in some spermatogonia of the neonatal testis. The functional significance of the latter findings remains open. The transgene was only expressed in adult Leydig cells; no expression was found in the fetal population of these cells. Hence, these findings indicate that the immediate 2-kb fragment of the murine LHR 5'-flanking sequence is transcriptionally active only in adult Leydig cells and certain brain areas, but other promoter sequences are apparently needed for ovarian and fetal testicular expression of the LHR gene.

Repopulation during radical radiotherapy for T1 glottic cancer.

BACKGROUND AND PURPOSE: Because opinions on the significance of repopulation during radiotherapy of T1 laryngeal cancer vary, we have estimated the effective rate of tumour cell repopulation during radiotherapy in patients with T1 laryngeal cancer. MATERIALS AND METHODS: One hundred seventeen consecutive patients with T1 laryngeal cancer were treated from 1982 to 1993 by radical radiotherapy alone either as continuous (n = 28) or split-course treatment (n = 89). The logit method of the linear-quadratic formula for local control at 3 years was used to examine the effect of treatment time on local control. The analysis was made for all patients to obtain a wide range of overall treatment times. RESULTS: The 3-year overall survival rate was 76% and the 3-year local control rate was 85% (range 82-88%). The local control rates were 95% (range 94-96%) for the continuous and 81% (range 75-91%) for the split-course therapy groups, respectively. The results showed a mean Dprolif value at the steepest part of the response versus time curve of 0.48 Gy/day for local control at 3 years although this was not statistically significant. The trade-off of dose required to compensate for a 1 week increase in treatment time for local control at the 90% level achieved at 3 years was calculated to be 3.5 Gy. CONCLUSIONS: The present results suggest that repopulation should be taken into account even when treating small T1 laryngeal cancer and that protraction of the overall treatment time should be avoided.

Striatal presynaptic dopamine function in type 1 alcoholics measured with positron emission tomography.

Recent in vivo studies have shown low dopamine D2 receptor and dopamine transporter densities among late onset (type 1) alcoholics. We have now studied 6-[18F]-FDOPA (FDOPA) uptake in 10 type 1 alcoholics and eight matched controls to test the hypothesis that striatal presynaptic dopamine function is lower among alcoholics. Markedly low FDOPA uptake (Ki) was observed in the left caudate of two alcoholic patients, but the mean striatal uptake values of the patient group were higher than those of the control group. The greatest difference was observed in the mean FDOPA intake in the left putamen, which was 28% higher in the patient group (t = 3.00, P = 0.008, d.f. = 16, independent samples t-test), and in the right caudate (difference 36%, t = 2.87, P = 0.01). The elevated FDOPA uptake in putamen and caudate correlated with poor Wisconsin Card Sorting Test (WCST) performance (error %) among alcoholics (correlation coefficients from 0.49 to 0.56), which suggests that the magnitude of presynaptic dopamine function alteration correlates with the degree of disability to modify one's behavior. The results do not give support to the hypothesis of generally decreased striatal dopamine turnover in type 1 alcoholism, but on the contrary indicate an increased presynaptic dopamine function. This may represent a compensatory mechanism to low postsynaptic DA function. The low presynaptic DA function observed in the left caudate of two patients suggests that type 1 alcoholism may be a heterogeneous disorder.

Transgenic mouse models for gonadal tumorigenesis.

The versatile transgenic (TG) techniques allow the production of in vivo animal models for a variety of diseases, including malignant tumors, through tissue-specific expression of oncogenes. We have created a TG mouse model for gonadal somatic cell tumors by expressing the powerful viral oncogene, Simian virus 40 T-antigen (Tag) under regulation of the murine inhibin alpha-subunit promoter (inh alpha). Ovarian granulosa and theca cell tumors were formed in the female, and those of testicular Leydig cells, in the male TG mice at the age of 5-6 months, with 100% penetrance. The tumors produced high levels of inhibin peptides, especially the alpha-subunit, and were steroidogenically active, mainly producing progesterone. The gonadal tumorigenesis was gonadotropin-dependent, since TG mice rendered gonadotropin-deficient by crossbreeding them into the hypogonadotropic hpg genetic background, or by treating them with a gonadotropin-releasing hormone (GnRH) antagonist, did not develop tumors. In order to study the possibility of using the tumor mouse model for testing gene therapy, we created another TG mouse model expressing under the same inhibin-alpha promoter the Herpes Simplex virus (HSV) thymidine kinase (TK) transgene. The inh alpha/HSV-TK mice were crossbred with the inh alpha/Tag mice and the double mutant mice also developed gonadal tumors. When they were treated with antiherpes drugs (acyclovir or gancyclovir), further growth of the tumors was blocked. These preliminary findings prove the principle that tumor ablation in our TG mouse model can be achieved by transduction of the HSV-TK gene into the tumor cells. Besides studies of formation, regulation and therapy of the tumors in vivo, immortalized cell lines derived from them provide models for studies of gonadal somatic cell functions in vitro.

Neurodevelopment until the adjusted age of 2 years in extremely low birth weight infants after early intervention--a case-control study.

A total of 104 infants with birth weights of less than 1000 grams were enrolled in this prospective case-control study in order to examine the effect of occupational therapy based on sensory integration (SI) and neurodevelopmental therapy (NDT) on neurological development. The children were grouped as matched pairs on the basis of a set of developmental risks assessed at the age of 3 months. The intervention children had a weekly session of 60 minutes of occupational therapy from the corrected age of 6 months up to 12 months. All the children were examined at the corrected age of 3, 6, 9, 12, 18 and 24 months. The neurodevelopment of the cases and the controls did not differ essentially and the only significant difference was found in the social development of the children at the age of 12 months to the advantage of the intervention group. It is concluded that this amount of occupational therapy in at-risk children does not have a relevant effect on neurological development.

Childhood family problems and current psychiatric problems among young violent and property offenders.

OBJECTIVE: The main objective was to examine whether young property and violent offenders would differ from each other in the prevalence of childhood abuse and neglect experiences, prevalence of various early family problems, and prevalence of disruptive behavior disorders, depression, and substance use. METHOD: Childhood abuse and neglect assessments and family problems were based on interview, questionnaire, and file data. Psychiatric diagnoses were made on the basis of a structured clinical interview. RESULTS: There were no statistically significant differences in the prevalence of childhood physical or psychological abuse, or neglect between the groups. According to the files, physical abuse was experienced by 57.5% of the violent offenders compared with 37% of the property offenders (p = .10). The prevalence rates of family problems were not different in the groups. Seventy-one percent of the violent group abused street drugs compared with 51% of the property group (p < .05), but the groups did not differ in disruptive behavior disorders, alcohol abuse/dependence, or depression. CONCLUSIONS: The property and violent offenders were surprisingly similar to each other in childhood experiences, family problems, and psychiatric diagnoses. The prevalence of childhood family and psychiatric problems was high in both groups.

Development of luteinizing hormone action in the perinatal rat ovary.

The expression of LH receptor (LHR) mRNA was studied in fetal rat gonads using polymerase chain reaction multiplication of reverse-transcribed mRNA. A primer pair corresponding to the extracellular domain of the receptor revealed the expression of LHR mRNA in fetal ovaries and testes as early as embryonic Day 13.5, the earliest age studied. The localization of LHR mRNA was examined in the perinatal rat ovary using in situ hybridization with two antisense probes, one encoding the extracellular and the other encoding the transmembrane domains of LHR. At the age of 4 days, only the extracellular LHR probe gave specific signal over ovarian stromal and follicular cells, excluding the ova. Three days later, similar distribution of specific hybridization was observed with both probes. In the 10- and 30-day-old rat ovaries, clear expression of LHR mRNA was found to be with both probes in theca cells. Gonadotropin-stimulated production of cAMP was studied in cultures of dispersed perinatal rat ovarian cells. When 5-day-old ovarian cells were cultured for 3 days in vitro and then stimulated by either hCG (0.1 mg/L) or FSH (1 mg/L) for 6 h, cAMP production was enhanced only in cells stimulated by FSH. In a similar experiment with 7-day-old ovarian cells, cAMP production was stimulated by both FSH and hCG. Stimulation with hCG (0.1 mg/L) during the 3-day culture caused homologous desensitization of cAMP production, but stimulation with FSH (0.1 mg/L) had no such effect. The desensitization of the LHR was also investigated by treating neonatal rats in vivo with a high dose of hCG (600 IU/kg BW s.c. as a single injection on Day 7) or with a dosage of recombinant human (rec) FSH on Days 3-9 (0.3 IU s.c twice daily). Thereafter, at the age of 10 days, the ovaries were incubated either with recFSH (200 IU/L) or hCG (CR-121; 0.1 mg/L) for 1 h. Homologous desensitization of cAMP production by hCG was observed, but the FSH-mediated cAMP production was not affected. The hCG-induced steroidogenesis (progesterone and testosterone production) was not desensitized. In conclusion, these findings indicate that 1) the expression of the mRNA encoding the extracellular domain of LHR, i.e., truncated receptor, occurs in fetal rat gonads as early as embryonic Day 13.5; 2) the expression of the truncated LHR mRNA occurs uniformly in the differentiating ovarian cells before the appearance of the functional theca cell layer; 3) full-length LHR message appears in the developing ovary concomitantly with appearance of differentiated theca cells; 4) homologous desensitization of cAMP output by hCG, without steroidogenic desensitization, is present in perinatal rat ovaries; and 5) no FSH-evoked desensitization of cAMP production occurs in perinatal ovaries.

The follicle-stimulating hormone (FSH) beta- and common alpha-subunits are expressed in mouse testis, as determined in wild-type mice and those transgenic for the FSH beta-subunit/herpes simplex virus thymidine kinase fusion gene.

Testicular expression of the endogenous FSH beta-subunit (FSH beta) and common alpha-subunit (C alpha) genes, as well as a Herpes simplex virus type 1 thymidine kinase (tk) transgene, driven by a 2.3-kilobase fragment of the bovine GSH beta promoter, were studied at messenger RNA and protein level in normal and transgenic mice. A major 3.8-kb species of FSH beta messenger RNA was demonstrated i the normal mouse testis by Northern hybridization. This was longer than the main 1.7-kb FSH beta transcript detected in the pituitary gland. Reverse transcription-polymerase chain reaction, followed by Southern hybridization, demonstrated FSH beta and tk expression in the pituitary gland and gonads of adult normal and transgenic mice, respectively. The C alpha expression was detected by reverse transcription-polymerase chain reaction in the pituitary gland and testis. During development, testicular transcription of the FSH beta and tk genes was initiated simultaneously a few days after birth. Immunocytochemistry of adult testes showed stage-specific positive reaction with FSH beta, C alpha, and tk antisera in the pachytene spermatocytes and type B spermatogonia, but not in Sertoli cells. Positive reaction with these antisera was also seen in the interstitial tissue. These results demonstrate testicular expression of the endogenous FSH subunit genes and confirm that the testicular expression of the FSH beta /tk transgene reflects or its subunits play a paracrine or autocrine role in the regulation of testicular function.

Ontogeny of luteinizing hormone receptor gene expression in the rat testis.

The ontogeny of expression of the LH receptor (LHR) gene was studied in rat testis between day 12.5 of fetal life and adulthood. Specific hybridization of testicular mRNA with a LHR cRNA probe encoding the extracellular domain of the receptor was found from day 16.5 of fetal life onward in Northern hybridization. Transcripts of 6.8, 4.2, and 2.7 kilobases were present at all ages, and a 1.8-kilobase species was present mainly in the adult testes. Hybridization was most intensive in day 21.5 fetuses, decreased after birth, and increased again by adulthood. The LHR mRNA was also analyzed by the reverse transcriptase-polymerase chain reaction technique, with primers multiplying either the full-length LHR mRNA or its extracellular domain. The specificity of the DNA species generated was verified by Southern hybridization using a nested 32P-labeled oligonucleotide. The results indicated that a truncated mRNA form, encoding the extracellular part of LHR, appears 1 day before the full-length LHR mRNA, i.e. on fetal days 14.5 and 15.5, respectively. This is in striking contrast to the rat fetal ovary, in which a difference of more than 10 days is found in the appearance of these two LHR mRNAs (17.5 days of fetal and 7 days of postnatal age, respectively). The appearance of the full-length LHR mRNA coincides in both sexes with the developmental onset of LHR binding observed in earlier studies. In situ hybridization using an antisense cRNA probe demonstrated that the LHR mRNA was confined to Leydig cells at all fetal and postnatal ages studied. In conclusion, there is good correlation in the developing rat testes between the onset of LHR gene expression and LHR binding, as observed in earlier studies. The findings in the fetal testis are at striking variance with those in the ovary, which starts expressing the extracellular domain of the LHR mRNA at roughly the same age as the testis. However, the appearance of full-length LHR mRNA and the functional receptor are delayed until day 7 postpartum.

The FSH beta-subunit promoter directs the expression of Herpes simplex virus type 1 thymidine kinase to the testis of transgenic mice.

The bovine FSH beta-subunit promoter (2.3 kb) was coupled to the coding sequence of the Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene and introduced into mouse embryos. A full-length tk transcript was found in the pituitary and testis. In the testis an additional truncated version of tk mRNA was also expressed. Two sets of primer extension fragments were identified, one corresponding to transcription initiation at or near the cap site of the FSH-beta gene, the other to transcription initiation within the tk gene. Furthermore, the latter, shorter transcript contained a 227 bp deletion. Only the long transcript was translated into immunoreactive tk in the later stages of developing spermatids. The tk protein was also functional in the testes, since spermatogenesis was either arrested or the germinal epithelium almost completely destroyed in transgenic males treated with the antiherpetic agent. If the FSH-beta-HSV-tk transgene also functions correspondingly in the pituitary, these mice will provide a useful model for studies on FSH.

Ontogeny of gonadotropin action in the rat ovary.

Specific binding of radiolabelled FSH and LH to rat ovaries was demonstrated at the age of 7 days. However, when the biological response to LH and FSH was monitored by cAMP production in vitro, the FSH response appeared earlier than that of LH, on days 4 and 7, respectively. Cholera toxin stimulated cAMP production even in fetal ovaries, suggesting the presence of functional post-receptor machinery of cAMP production. Hence, the appearance of the functional gonadotropin receptor probably plays a key role in the onset of postnatal ovarian steroidogenesis. To test the effect of gonadotropin suppression during postnatal ovarian development, a potent GnRH antagonist was administered to neonatal animals between days 1-6 or 1-9 of life. The ovarian responsiveness to FSH developed even in the absence of normal gonadotropin levels, but that to LH was suppressed after the longer antagonist treatment. The temporal relationship between the onset of LHR gene expression, i.e. transcription, and translation to functional receptor protein was thereafter investigated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The measurements revealed the existence only of truncated versions of LHR mRNA in the fetal ovary from day 17 of gestation up to day 7 of postnatal life. With the onset of the receptor function around day 7, also larger mRNA transcripts, corresponding to the full-length receptor protein appeared. Our findings suggest that the LHR gene may be constitutively expressed in the ovary and a change in the alternative splicing pattern may cause the onset of translation of a functional receptor protein.

Functional LH receptor appears in the neonatal rat ovary after changes in the alternative splicing pattern of the LH receptor mRNA.

The LH receptor (R) function, as assessed by ligand binding and stimulation of cAMP production, is initiated in the neonatal rat ovary around day 7 of life. To correlate this with the onset of expression of the LHR gene, we assessed the presence of the cognate mRNA in the developing rat ovary from day 17 of fetal life onwards using PCR multiplication of reverse transcribed LHR mRNA, followed by southern hybridization and sequencing of the PCR products. Our findings indicate that only truncated versions of LHR mRNA are present in the developing rat ovary as early as day 17 fetal life and up to day 7 post partum. Concomitant with the appearance of a functional LH receptor (after day 7 post partum), two larger LHR mRNA species (2.6 and 6.7 kb) appear, which are also present in the adult ovary. These findings indicate that the LHR gene is probably constitutively expressed in the developing rat ovary, and that the onset of translation of functional LH receptor occurs through a change in the alternative splicing pattern of LHR mRNA.

Amplification of the hypervariable region close to the apolipoprotein B gene: application to forensic problems.

Our purpose was to identify individuals from blood stains in two murder cases. We used primers flanking the hypervariable region of the apoB gene to amplify DNA extracted from blood stains and blood samples from suspected persons. The sensitivity and specificity of the procedure was improved by carrying out two consecutive PCR amplifications with a nested set of primers in the second amplification. The size of the generated fragments was determined by polyacrylamide gel electrophoresis followed by staining with ethidium bromide. By comparing the fragments produced from the stains with those from the blood samples we were able to identify the origin of the blood stains in both cases.

Absence of familial defective apolipoprotein B-100 in Finnish patients with elevated serum cholesterol.

Familial defective apolipoprotein B-100 is a genetic disorder which is associated with elevated plasma LDL levels. It appears to result from a G----A mutation at nucleotide 10,708 in exon 26 of the apolipoprotein B-100 gene leading to a substitution of glutamine for arginine at amino acid residue 3500. We explored the possible role of this point mutation as a cause of elevated plasma cholesterol among the Finns, a genetically isolated population in which both hypercholesterolemia and coronary heart disease are common: 552 hyperlipidemic patients from Western and Southern Finland were screened either by assaying patient sera with monoclonal antibody MB47 or by amplifying the region of the apo B gene containing the nucleotide 10,708 followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes. Not a single individual with this particular mutation could be found. We conclude that familial defective apo B-100 is not a common cause of elevated plasma cholesterol in this population.

XbaI polymorphism of the apolipoprotein B gene influences plasma lipid response to diet intervention.

Fresh blood samples were collected from 103 North Karelians who had in 1981-84 participated in dietary intervention studies and analysis of the XbaI restriction fragment length polymorphism (RFLP) of apolipoprotein B (apoB) was carried out. Reanalysis of the original plasma lipid and apolipoprotein data indicated that while baseline concentrations did not differ significantly between genotypes, the response to a low-fat, low-cholesterol diet was influenced by apoB XbaI genotype: reductions in total and low density lipoprotein (LDL) cholesterol, apoB and high density lipoprotein (HDL) cholesterol were greater in subjects homo- or heterozygous for the presence of the XbaI cutting site (X1X2 or X2X2 genotype, designated X2+) as compared to those lacking the cutting site (X1X1 genotype, designated X2-). The corresponding average reductions induced by dietary intervention in X2+ and X2- subjects were: for total cholesterol 1.30 and 0.99 mmol/l (p = 0.036), for LDL cholesterol 1.04 and 0.78 mmol/l (p = 0.049), for apoB 18.3 and 8.1 mg/100 ml (p = 0.012) and for HDL cholesterol 0.26 and 0.17 mmol/l (p = 0.008).