Serum microRNAs as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

Association between a polymorphism affecting an AP1 binding site in the promoter of the TCIRG1 gene and bone mass in women.

The TCIRG1 gene encodes a component of the osteoclast vacuolar proton pump and previous work has shown that inactivating mutations of the TCIRG1 cause autosomal recessive osteopetrosis. In order to determine whether allelic variation in TCIRG1 contributes to the regulation of bone mineral density (BMD) in normal individuals, we studied the relationship between polymorphisms of TCIRG1 and BMD in a population-based cohort of 739 perimenopausal women. Five common polymorphisms were identified: two in the promoter, a conservative change within exon 4, one within intron 4 and one within intron 11. One of the promoter polymorphisms (G-1102A) lay within a consensus recognition site for the AP1 transcription factor. There was a significant association between the G-1102A genotype and BMD at the lumbar spine ( P = 0.01) and femoral neck ( P = 0.03). The association remained significant after correcting for age, weight, height, menopausal status/HRT use and smoking ( P = 0.008 for spine BMD and P = 0.03 for hip BMD), and homozygotes for the -1100 "G" allele had BMD values significantly higher than individuals who carried the -1100 "A" allele at both spine ( P = 0.007) and hip ( P = 0.047). Subgroup analysis showed that the association between G-1102A and BMD was restricted to premenopausal women who comprised 50.6% of the study group. None of the other polymorphisms or haplotypes were significantly associated with BMD in the study group as a whole or in any subgroup. Functional studies will need to be performed to determine the mechanisms that underlie this association, but we conclude that, in this relatively large population, allelic variation at the G-1102A site of TCIRG1 accounts for part of the heritable component of BMD in Scottish women, possibly by affecting peak bone mass.

The mutational spectrum of human malignant autosomal recessive osteopetrosis.

Human malignant infantile osteopetrosis (arOP; MIM 259700) is a genetically heterogeneous autosomal recessive disorder of bone metabolism, which, if untreated, has a fatal outcome. Our group, as well as others, have recently identified mutations in the ATP6i (TCIRG1) gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, are responsible for a subset of this condition. By sequencing the ATP6i gene in arOP patients from 44 unrelated families with a worldwide distribution we have now established that ATP6i mutations are responsible for approximately 50% of patients affected by this disease. The vast majority of these mutations (40 out of 42 alleles, including seven deletions, two insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to cause severe abnormalities in the protein product and are likely to represent null alleles. In addition, we have also analysed nine unrelated arOP patients from Costa Rica, where this disease is apparently much more frequent than elsewhere. All nine Costa Rican patients bore either or both of two missense mutations (G405R and R444L) in amino acid residues which are evolutionarily conserved from yeast to humans. The identification of ATP6i gene mutations in two families allowed us for the first time to perform prenatal diagnosis: both fetuses were predicted not to be affected and two healthy babies were born. This study contributes to the determination of genetic heterogeneity of arOP and allows further delineation of the other genetic defects causing this severe condition.

Effects of ECS on DNA single-strand breaks in rat brain cells.

We conducted a series of experiments to evaluate possible molecular mechanisms by which electroconvulsive therapy, a commonly used treatment for depression, may exert its adverse effects such as amnesia. We assessed the effects of repeated electroconvulsive shocks (ECS) alone and in combination with low-level radiograph irradiation on DNA single-strand breaks in cells in the rat brain, using a sensitive alkaline microgel electrophoresis assay method. Our results show that ECS, when administered alone, had no significant effects on DNA single-strand breaks in cells in either the hippocampus or the rest of the brain. However, repeated ECS when combined with a low-level radiograph irradiation produced a small but significant increase in DNA single-strand breaks in rat brain cells.

Effects of low-dose TRH on cognitive deficits in the ECT postictal state.

A low dose (0.5 mg) of thyrotropin-releasing hormone (TRH), a short-acting tripeptide with known analeptic properties, was administered to eight depressed patients 5 minutes after ECT session 3 or 4 in a double-blind, placebo-controlled crossover design. After TRH infusion the patients displayed selectively better performance on a battery of neuropsychological tests than they did after placebo infusion. Further exploration with pharmacological probes to mitigate ECT postictal cognitive deficits is warranted.

NS-3, a TRH analog, reverses repeated ECS-induced deficits in water maze performance in the rat.

Rats given five consecutive daily electroconvulsive shock (ECS) treatments and trained to run in the Morris water maze, starting three days posttreatment, showed deficits in learning and memory functions. Treatment before each training session with the thyrotropin-releasing hormone (TRH) analog NS-3 [(CG-3703), (3R),(6R)-6-methyl-5-oxo-3-thiomorphorinyl-l-histidyl-l-prolinamid e tetrahydrate] reversed these behavioral deficits. The possible use of TRH and its analogs as therapeutic treatment for the cognitive dysfunctions resulting from electroconvulsive shock treatment for depression and the possible involvement of central cholinergic systems in the cognitive dysfunctions are discussed.

Can ECT-induced cognitive effects be altered pharmacologically?

1. A systematic review of the literature revealed twelve clinical trials that evaluated nine different drugs, and used three different conceptual models to prevent, restore or treat ECT-induced cognitive deficits. 2. This review indicated inconclusive results regarding clinical utility of any of the drugs. 3. Major factors discussed include the complexities involved in the evaluation of ECT-induced cognitive deficits, and the techniques of evaluating changes in cognitive functions. 4. Our conclusion is that future research should emphasize understanding the neural mechanisms related to ECT-induced cognitive deficits. We suggest several areas for future exploration.

Electroconvulsive therapy.

Modern ECT practice has evolved far from its beginnings more than 50 years ago. ECT is effective, safe, and rewarding in the clinical setting. This discussion complements the 1990 APA Task Force report and elaborates on some of the clinical and scientific factors that could not be fully addressed by the report. The future of ECT lies in understanding the mechanisms by which it relieves depressive symptoms and causes amnesia and related cognitive deficits.

Low-dose thyrotropin-releasing hormone effects in cognitively impaired alcoholics.

The cognitive effects of a low dose of thyrotropin-releasing hormone (TRH) (2.0 mg, IV) were evaluated in 18 chronic alcoholic patients who exhibited memory dysfunction secondary to chronic alcohol abuse. The study used a double-blind crossover design that compared cognitive functions in patients with 2.0 mg of TRH IV as compared with a placebo. TRH was chosen because of its ability to enhance cholinergic transmission. Only minimal effects were seen with TRH. Patients with a shorter duration of alcohol use (mean of 16 years) performed significantly better with TRH as compared with placebo on a test involving verbal learning and memory. Those with a more chronic history of alcohol abuse (mean of 27 years) did not show such a response. All of the subjects showed cardiovascular response to TRH. Factors that may have contributed to the results of our study are discussed. It is our impression that future studies evaluating the cognitive effects of TRH in chronic alcoholics need to include an evaluation of the functional activity of TRH in the brain.

Depression in the elderly: a treatable disorder.

Up to 15% of elderly women may suffer from depression. Patients with this disorder present with change in mood or diminished interest or pleasure in usual activities, as well as a variety of other neurovegetative symptoms. Depression is amenable to treatment, and a wide range of drug and nonpharmacologic modalities may be useful. The mere action of seeking professional help signals that the depressed patient has begun to take control of the situation and serves as a promising sign for recovery. The tricyclic antidepressants have a long history of use for the treatment of depression and are probably the most widely used agents for this indication, despite numerous adverse effects. Bupropion and the new selective serotonin reuptake inhibitors are effective in treating depression and are better tolerated than the tricyclics. For this reason, they may be particularly useful for treating depression in the elderly.

ECT and TRH: cholinergic involvement in a cognitive deficit state.

In this paper we describe the findings from two preliminary experiments, a human and an animal study, investigating whether thyrotropin-releasing hormone (TRH) can mitigate electroconvulsive therapy (ECT)-induced cognitive deficits. Our results suggest further explorations of TRH and its analogs as possible therapeutic agents for these deficits. We speculate that the major cause of the ECT-induced cognitive deficits is a decrease in cholinergic transmission in the central nervous system. Treatments such as TRH, which enhance cholinergic activity, can reverse the cognitive deficits.

Persistence of HIV-1 silent infection in seronegative subjects at high risk.

Twenty regular sexual partners of HIV-1 infected subjects, without detectable human immunodeficiency virus (HIV-1) antibody and positive for HIV-1 genome by in situ hybridization (ISH), were selected and studied longitudinally for 6-36 months to estimate the duration of silent infection. During the follow-up period, 10 showed atypical Western Blot (WB) patterns. Two seronegative partners seroconverted. Rapid progress to AIDS was observed in 7 seropositive subjects.

HIV genome in peripheral blood mononuclear cells of seronegative regular sexual partners of HIV-infected subjects.

We have investigated the presence of the human immunodeficiency virus (HIV) by using in situ hybridization on peripheral blood mononuclear cells (PBMCs) from seronegative regular sexual partners of HIV-infected subjects. The cells were hybridized with a 9 kilobase (kb) Sstl-Sstl lambda BH 10 probe, which was able to recognize both viral mRNA and proviral cDNA. Labeling was done by chemical insertion of an antigenic sulfone group in cytosine moieties and was visualized by a double-antibody immunohistochemical reaction. In all the subjects studied, the HIV genome was present. The HIV infected cells showed morphological aspects consistent with that of lymphocytes and monocytes. Our data suggest that the anti-HIV seronegative individuals who are regular sexual partners of HIV-infected subjects may be HIV-infected.

HIV viral sequences in seronegative people at risk detected by in situ hybridisation and polymerase chain reaction.

A study was conducted to assess the occurrence of latent infection with the human immunodeficiency virus (HIV) among seronegative people at high risk of infection. The presence of HIV genomes was analysed by molecular techniques in two seronegative children born to mothers infected with HIV and in three regular sexual partners of seropositive drug addicts. The adults were selected from a seronegative cohort at high risk of infection because of their sexual contacts and the children selected because of impaired growth. HIV retroviral sequences were detected in four of the five subjects directly at the cellular level by in situ hybridisation in peripheral blood mononuclear cells. HIV genomic sequences were confirmed by in vitro amplification of viral DNA with the polymerase chain reaction technique. The existence of a latent viral infection state in these seronegative subjects indicates the unreliability of standard serological analysis in people who have been in regular contact with infected patients.

Immunohistochemical demonstration of p24 HTLV III major core protein in different cell types within lymph nodes from patients with lymphadenopathy syndrome (LAS).

The p24 protein is the major core protein of LAV/HTLV III which is the putative agent of the lymphadenopathy syndrome. By the use of an anti-p24 monoclonal antibody we have studied the presence of LAV/HTLV III infected cells in 20 lymph nodes obtained from lymphadenopathy syndrome patients: 14 lymph nodes were characterized by prominent follicular hyperplasia consistent with the early phase of the syndrome and six lymph nodes presented marked regressive changes. Cells positive for p24 were detected in 8/14 lymph nodes with hyperplastic changes and in 1/6 lymph nodes with regressive changes. Positive cells were most often located in germinal centres and were mainly characterized by a lymphoid morphology. However, immunoreactivity for p24 protein was also occasionally observed in some histiocytic-like cells and in high endothelial cells of post-capillary venules, suggesting that these 'accessory cells' also play a role in the early phases of the lymphadenopathy syndrome.

Systemic lymphadenopathy (LAS) in intravenous drug abusers. Histology, immunohistochemistry and electron microscopy: pathogenic correlations.

In the present study we have evaluated the histological, immunohistochemical, ultrastructural and cell surface phenotypic features of lymph nodes from 19 intravenous drug abusers and two homosexual men with persistent lymphadenopathy syndrome and from six control patients. Our investigation has demonstrated that the lymphadenopathy of drug abusers is characterized by: (a) specific histological features with evidence of evolution from an initial hyperplastic-reactive to a late regressive-destructive stage; (b) T-cell surface phenotype distribution similar to that in the peripheral blood of homosexuals; (c) peculiar infiltration of the germinal centres by T8+ cells and by S-100+, T6+ interdigitating reticulum-like cells; (d) electron microscopic features indicating the presence of virus bodies in lymph node cells and in the intercellular spaces. These data suggest that the lymphadenopathy of drug abusers and homosexuals are similar pathological conditions with characteristic features which allow histological differentiation of this entity from those found in other viral infections.

The Omenn's syndrome: histological, immunohistochemical and ultrastructural evidence for a partial T cell deficiency evolving in an abnormal proliferation of T lymphocytes and S-100 +/T-6 + Langerhans-like cells.

A 7 month old female infant was affected by a rapidly fatal familial disease highly reminiscent of Omenn's syndrome. She presented with widespread eczematous lesions, hepatosplenomegaly, superficial lymphadenopathy, peripheral blood lymphocytosis, eosinophilia and hyper-IgE. An axillary lymph node was involved by a marked proliferation of T-3 +/T-10-- lymphocytes admixed with S-100+/T-6+/Leu-3a+/Ia + reticular cells which lacked typical LC granules; cell suspension study revealed that 90%-96% of the lymph node cells were T-11+/T-3+ lymphocytes characterized by low expression of Leu-3a and T-8 antigens and by high expression of Ia antigens (52%). Peripheral blood T lymphocytes exhibited a similar distribution of surface phenotypes. The patient died of interstitial pneumonia and an autopsy was performed. The thymus was markedly atrophic and completely devoid of lymphocytes. The peri-arteriolar lymphoid sheets of the spleen were poorly developed and were mainly composed of T-8+ lymphocytes. The mediastinal nodes were rudimentary and were populated by T-3+/T-10+ lymphocytes with low expression of Leu-3a and T-8 antigens. Our results raise the possibility that Omenn's syndrome is a peculiar primary immunodeficiency in which, despite early thymic involution, some abnormal T lymphocytes still develop in the peripheral lymphoid organs. Antigenic triggering of these cells might result in prominent proliferations of T lymphocytes and Langerhans-like cells which lead to the clinical manifestation of the disease.

Peripheral T cell lymphoma in adults: morphological and phenotypical study of four cases.

In the present study we investigated the lymph node morphology and distribution of cell surface phenotypes in four cases of adult peripheral T cell lymphoma. Histologically, the tumors were classified as T zone lymphoma, T cell lymphoma with large multilobated nuclei and T cell immunoblastic sarcoma. In the T zone lymphoma the neoplastic lymphocytes were E+ (90%) and exhibited intensive focal staining for acid phosphatase (93%) and acid esterase (92%); the phenotype distribution revealed low expression of the T-3 antigen (49%), selective expression of the T-4 antigen (72%) and poor expression of T-6 (10%) and T-10 antigens (22%). Some of these features are present in normal and in neoplastic immature T cells. In the remaining three cases the majority of lymph node cells were E+ (59-75%), T-3+ (67-80%) and T-8+ (43-55%). A distinctive feature of the T cell immunoblastic sarcoma was the presence of high percentages of DR+ cells (62%; 63%). Thus our results indicate that the morphological heterogeneity of peripheral T cell lymphoma is also paralleled by a variety of surface phenotypes and that phenotype studies may provide a useful contribution to identification and accurate classification of peripheral T cell neoplasms.