Obesity and obesogenic growth are both highly heritable and modified by diet in a nonhuman primate model, the African green monkey (Chlorocebus aethiops sabaeus).

OBJECTIVE: In humans, the ontogeny of obesity throughout the life course and the genetics underlying it has been historically difficult to study. We compared, in a non-human primate model, the lifelong growth trajectories of obese and non-obese adults to assess the heritability of and map potential genomic regions implicated in growth and obesity. STUDY POPULATION: A total of 905 African green monkeys, or vervets (Chlorocebus aethiops sabaeus) (472 females, 433 males) from a pedigreed captive colony. METHODS: We measured fasted body weight (BW), crown-to-rump length (CRL), body-mass index (BMI) and waist circumference (WC) from 2000 to 2015. We used a longitudinal clustering algorithm to detect obesogenic growth, and logistic growth curves implemented in nonlinear mixed effects models to estimate three growth parameters. We used maximum likelihood variance decomposition methods to estimate the genetic contributions to obesity-related traits and growth parameters, including a test for the effects of a calorie-restricted dietary intervention. We used multipoint linkage analysis to map implicated genomic regions. RESULTS: All measurements were significantly influenced by sex, and with the exception of WC, also influenced by maternal and post-natal diet. Chronic obesity outcomes were significantly associated with a pattern of extended growth duration with slow growth rates for BW. After accounting for environmental influences, all measurements were found to have a significant genetic component to variability. Linkage analysis revealed several regions suggested to be linked to obesity-related traits that are also implicated in human obesity and metabolic disorders. CONCLUSIONS: As in humans, growth patterns in vervets have a significant impact on adult obesity and are largely under genetic control with some evidence for maternal and dietary programming. These results largely mirror findings from human research, but reflect shorter developmental periods, suggesting that the vervet offers a strong genetic model for elucidating the ontogeny of human obesity.

Adult age confounds estimates of static allometric slopes in a vertebrate.

In many animal groups, the size of male genitalia scales shallowly with individual body size. This widespread pattern appears to admit some exceptions. For instance, steep allometries have been reported for vertebrate genitalia. This exception, however, may be due to a confounding effect arising from the continued growth of some structures during adulthood in vertebrates. Consider the possibility that genitalia continue to grow in adults while body size does not. If so, taking measurements from adults of different ages could yield steeper allometries than would be obtained from measurements of adults of the same age. We used vervet monkeys to test this hypothesis. We found that all body parts continued to grow in adult vervet monkeys, with sexual traits (including genitalia) showing faster growth rates. Traits with faster growth rates over adult ages had steeper allometries. And accounting for variation in adult age yielded shallower allometries, bringing vervet monkey genitalia in line with the predominant pattern observed in other animal groups. These results suggest that steep allometric slope estimates reported for other vertebrates may be due in part to mixing of adult ages, and reinforces one of the most consistent patterns yet detected in the study of static allometry.

Understanding homologous recombination repair deficiency in biliary tract cancers: clinical implications and correlation with platinum sensitivity.

BACKGROUND: Biliary tract cancers (BTCs) exhibit high mortality rates and significant heterogeneity in both clinical and molecular characteristics. This study aims to molecularly characterize a cohort of patients with BTC, with a specific focus on genomic alterations within homologous recombination repair (HRR) genes in a real-world setting. PATIENTS AND METHODS: We carried out a retrospective analysis on 256 patients with BTC treated at five Austrian centers and one German comprehensive cancer center between 2016 and 2023 utilizing comprehensive genomic profiling platforms to assess HRR status and its correlation with clinical outcomes after platinum-based chemotherapy. RESULTS: A total of 67 patients (27.5%) exhibited HRR gene mutations (HRRm), with the most common pathogenic alterations in BAP1 (9%), ARID1A (7.8%), and ATM (6.1%). Time to failure of the first-line strategy (TFS) between patients with HRRm and non-HRRm treated with platinum agents was 7.9 and 6.7 months, respectively [hazard ratio (HR) 0.89; P = 0.49]. The overall survival (OS) estimates at 6, 18, and 24 months were 82%, 45%, and 39% in the HRRm group (median 16.01 months) and 81%, 42%, and 22% in the HRR group (median 15.68 months), respectively (Fleming-Harrington test P = 0.0004; log-rank P = 0.022). Significance did not persist in the multivariate analysis (HR 0.72; 95% confidence interval 0.489-1.059; P = 0.095). An interaction between HRRm status and molecular-informed therapeutic strategies in later lines was noted. In the second-line treatment, OS following an irinotecan-based regimen was comparable to re-exposure to platinum-based agents (12.36 versus 10.13 months; HR 0.92; P = 0.85). No better outcome was noted for patients with HRRm versus patients with non-HRRm with second-line platinum agents (HR 1.45; P = 0.35). CONCLUSIONS: Patients with HRRm with BTC showed a potential advantage in OS following platinum-based first-line chemotherapy, presumably attributed to enhanced opportunities for targetable coalterations. Further investigation is needed to outline HRR within the scope of BTCs and detail a clinically meaningful sensitivity to platinum agents or targeted approaches with poly (ADP-ribose) polymerase (PARP) inhibitors.

Genetic analysis of chemoresistance in primary murine lymphomas.

Understanding the basis of chemoresistance is a principal goal of molecular oncology. We have exploited a murine lymphoma model and retroviral gene transfer to rapidly generate a series of spontaneous tumors differing only in a gene of interest, and subsequently studied the impact of the test gene on the treatment sensitivity of tumors at their natural site. We demonstrate that the Bcl-2 oncoprotein produces multi-drug resistance when assessed in primary lymphomas in vivo. In contrast, this effect was dramatically reduced when the primary lymphomas were subjected to long-term culture, and completely missed in the standard clonogenic survival assay. This model highlights the importance of physiological test systems to address the complexity of clinical drug resistance and provides a novel strategy to evaluate compounds targeting specific genetic lesions.

Cellular senescence predicts treatment outcome in metastasised colorectal cancer.

BACKGROUND: Cellular senescence is a terminal cell-cycle arrest that occurs in response to activated oncogenes and DNA-damaging chemotherapy. Whether cancer cell senescence at diagnosis might be predictive for treatment outcome is unknown. METHODS: A senescence index (SI) was developed and used to retrospectively correlate the treatment outcome of 30 UICC stage IV colorectal cancer (CRC) patients with their SI at diagnosis. RESULTS: 5-Fluorouracil/leucovorin-treated CRC patients achieved a significantly longer progression-free survival when presenting with SI-positive tumours before therapy (median 12.0 vs 6.0 months; P=0.044). CONCLUSION: Cancer cell senescence predicts treatment outcome in metastasised CRC. Prospective analyses of larger patient cohorts are needed.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

Expression and regulation by interferon-gamma of the membrane-bound complement regulators CD46 (MCP), CD55 (DAF) and CD59 in gastrointestinal tumours.

The membrane-bound complement inhibitors CD46 (membrane cofactor protein), CD55 (decay-accelerating factor) and CD59 (protectin) protect tumour cells against lysis by activated complement. In this study, a total of 14 (3 gastric, 3 colonic and 8 pancreatic) gastrointestinal tumour cell lines were examined for the expression of CD46, CD55 and CD59 with respect to the regulatory efficacy of interferon-gamma (IFN-gamma). The effects of IFN-gamma on mRNA and protein expression levels of CD46, CD55 and CD59 were evaluated by Northern blot hybridisation, RT-PCR, flow cytometry and immunostaining. In unstimulated cell lines, CD46 and CD59 transcripts were expressed at comparable levels, whereas the basal expression of CD55 mRNA was heterogeneous. The complement inhibitor proteins were detected in all cell lines using specific antibodies. Additional immunohistochemical stainings of gastrointestinal tissue specimens supported these findings. IFN-gamma evoked a weak induction of certain transcripts in a subset of the cell lines. Upregulation of protein expression was only observed in HT29 cells for CD55 and CD59 and was accompanied by a marked increase of the corresponding transcripts. We conclude that membrane-bound complement inhibitors are broadly expressed in gastrointestinal tumour cells and vary in their susceptibility to IFN-gamma. Thus, they may be involved in tumour escape mechanisms in gastric, pancreatic and colorectal cancer.

Norepinephrine transmitter metabolite induces apoptosis in differentiated rat pheochromocytoma cells.

3,4-Dihydroxyphenylglycolaldehyde (DOPEGAL) is the monoamine oxidase A metabolite of norepinephrine and epinephrine. DOPEGAL, but not other metabolites, kills differentiated PC-12 cells. However, the type of DOPEGAL induced cell death, whether necrosis or apoptosis, is not known. To determine the type of cell death triggered by DOPEGAL, PC-12 cells cultured in the presence or absence of 30 microM DOPEGAL were examined by electron microscopy and DNA agarose gel electrophoresis for characteristic features of apoptosis. Results indicate that DOPEGAL induces apoptosis in these cells. Implications for degenerative diseases are discussed.

Norepinephrine transmitter metabolite is a selective cell death messenger in differentiated rat pheochromocytoma cells.

3,4-Dihydroxyphenylglycolaldehyde (DOPEGAL) is the monoamine oxidase A (MAO-A) metabolite of norepinephrine (NE) and epinephrine (Epi). Oxidative metabolites of amines are predicted toxins. In this study we determine the toxicity of DOPEGAL, its tautomer 2',3,4-trihydroxyacetophenone (THAP) as well as NE, Epi and their oxidative and methylated metabolites in cultures of differentiated PC-12 cells. At 59.5 microM DOPEGAL, THAP and Epi, but not NE or other NE or Epi metabolites decreased PC-12 cells by 43.8%, 26.7% and 16.8% respectively. DOPEGAL toxicity was concentration and time dependent. Possible implications for degenerative diseases are discussed.

Accumulation of 3,4-dihydroxyphenylglycolaldehyde, the neurotoxic monoamine oxidase A metabolite of norepinephrine, in locus ceruleus cell bodies in Alzheimer's disease: mechanism of neuron death.

3,4-Dihydroxyphenylglycolaldehyde (DOPEGAL) is the neurotoxic monoamine oxidase A (MAO-A) metabolite of norepinephrine (NE). NE neurons in the locus ceruleus (LC) die in Alzheimer's disease (AD). To determine if DOPEGAL could contribute to NE neuron death in AD we measured levels of DOPEGAL, NE and their synthesizing enzymes in LC from AD and matched controls. We found 2.8- and 3.6-fold increases in DOPEGAL and MAO-A in AD LC neuronal cell bodies compared to controls. NE and dopamine beta-hydroxylase were increased by 3.8- and 10.7-fold, respectively. Implications for the mechanism of neuron death in AD are discussed.

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain.

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.

Developmental and thyroid hormone regulation of two molecular forms of Na+-K+-ATPase in brain.

In the brain there are two isozymes of Na+-K+-ATPase differing in their catalytic subunits: alpha, indistinguishable from the kidney form of alpha, and alpha +, found in axolemma. The time course of the increase in each alpha during development was described by quantitating the abundance of each form, studied in unpurified membranes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with specific antibodies and with fluorescein 5'-isothiocyanate. Both the alpha and alpha + subunits, quantitated with antibodies, increased 10-fold in abundance from 18 days gestation to 20 days of age, with alpha + increasing more rapidly than alpha early in development. A 10-fold increase in enzyme activity was also observed during this period. Using fluorescein 5'-isothiocyanate to quantitate the two alpha subunits, a similar increase in alpha + was observed with less of an increase in alpha. The ratio of alpha + to alpha increased from 0.75 at 18 days gestation to 3 at 3 days of age remaining at this ratio to 20 days of age. The possibility that thyroid hormone, a known regulator of brain Na+-K+-ATPase during development, differentially regulated the two forms was tested using 15-day-old hypothyroid rats. The abundance of both forms of alpha was similarly decreased: alpha + to 69% and alpha to 48% of control values. Na+-K+-ATPase activity was 70% of control. We conclude that both alpha and alpha + abundance increase in the brain during pre-and neonatal development and that the increase in both alpha subunits is regulated, directly or indirectly, by thyroid hormones.

Isozymes of dog heart Na+-K+-ATPase are immunologically similar to isozymes in brain.

The sodium pump, Na+-K+-ATPase, possesses two populations of cardiac glycoside-binding sites in cardiac tissue. This has been observed in other tissues such as brain where the two sites have been assigned to isozymes of Na+-K+-ATPase termed alpha and alpha +. In a previous study [Am. J. Physiol. 248 (Cell Physiol. 17): C247-C251, 1985], we were unable to demonstrate the presence of an alpha +-form in guinea pig heart sarcolemmal membranes using antibody probes. In the present study, using similar methodology, we show that dog, but not rat or guinea pig, sarcolemmal membranes contain two immunologically distinct alpha-subunits. The antibody-binding characteristics of dog heart alpha and alpha + are similar to the forms found in brain. The relative abundance of the two isozymes, estimated by labeling the sarcolemmal membranes with fluorescein 5'-isothiocyanate, was about equal. We conclude that the two populations of ouabain-binding sites in dog heart may result, at least in part, from the presence of the two isozymes of Na+-K+-ATPase in cardiac tissue of this species.

Apoptosis and therapy.

The dogma that antineoplastic treatments kill tumour cells by damaging essential biological functions has been countered by the notion that treatment itself initiates a programmed cellular response. This response often produces the morphological features of apoptosis and is determined by a network of proliferation and survival genes, some of which are differentially expressed in normal and malignant cells. Correspondingly, mutations that interfere with the initiation or execution of apoptosis may produce tumour-cell drug resistance. Remarkably, many of the genes that modulate apoptosis in response to cytotoxic drugs also affect apoptosis during tumour development; hence, the process of apoptosis provides a conceptual framework for understanding how cancer genes can influence the outcome of cancer therapy. Although the relative contribution of apoptosis to radiation and drug-induced cell death remains controversial, clinical studies have associated anti-apoptotic mutations with treatment failure. While careful preclinical and clinical studies will be necessary to resolve this point, our current understanding of apoptosis should facilitate the design of rational new therapies.

Bcl-2 mediates chemoresistance in matched pairs of primary E(mu)-myc lymphomas in vivo.

The oncoprotein Bcl-2 is a potent survival factor antagonizing p53-dependent and -independent apoptotic cell death. Although many anticancer agents are known to engage apoptotic pathways, the clinical impact of Bcl-2 on treatment outcome remains controversial. Since it might be difficult to assess the contribution of a single gene to treatment response in patient material due to technical considerations, we sought to address Bcl-2's role in a mouse model of primary lymphomas treated at their natural site. Driven by the E(mu)-enhancer controlled c-myc transgene, primary B cell lymphomas arise in this model by several months of age and resemble closely typical clinical and histopathological features of human non-Hodgkin lymphomas. We introduced either bcl-2 or a control construct into identical samples of freshly isolated E(mu)-myc lymphomas by retroviral gene transfer in order to obtain matched pairs of primary lymphomas differing only in their Bcl-2 status. While no Bcl-2-mediated effect was detectable in clonogenic survival assays in vitro, treatment of the genetically modified lymphoma pairs propagated in nontransgenic recipient mice revealed Bcl-2's impact on drug sensitivity in vivo. Bcl-2 efficiently blocked short- and long-term drug-mediated cell death in vivo. In a comparison of 15 matched pairs of primary lymphomas, the bcl-2 transduced sample never achieved longer remission periods than the control counterpart and most of the Bcl-2 overexpressing lymphomas failed to respond at all. We conclude that-when assessed in the physiological environmental context-MBcl-2 contributes to chemoresistance of B cell lymphomas in vivo. This model, able to test any other candidate gene, will be particularly useful to study the implications of specific mutations for drug action in vivo.

Clue cells in bacterial vaginosis: immunofluorescent identification of the adherent gram-negative bacteria as Gardnerella vaginalis.

Clue cells are epithelial cells covered by adherent gram-negative rods, observed in vaginal smears from women with bacterial vaginosis. Immunofluorescence studies were used to identify the gram-negative bacteria adhering to clue cells. Specific antisera to four common gram-negative vaginal bacteria (Gardnerella, Bacteroides, Fusobacterium, and Mobiluncus) were prepared by long-term, multiple, small-inoculum immunization of rabbits. Cross-reactivity with heterologous common vaginal bacteria was removed by absorption against whole cells of heterologous bacteria and by serial dilution. Gardnerella vaginalis was most often observed adhering to the surface of clue cells and was detected on the surface of exfoliated vaginal epithelial cells significantly more frequently and in higher numbers than were Mobiluncus, Bacteroides, and Fusobacterium, suggesting that this species of gram-negative bacteria is responsible for clue cell formation.

Blood pressure regulation in Alzheimer's disease.

Brain neurons which regulate blood pressure (BP), including the C-1 tonic vasomotor neurons, degenerate in Alzheimer's disease (AD). This study determines whether BP is decreased in AD. We reviewed records of three autopsy proven AD patients. Medical causes for decreased BP were investigated. Yearly averages for systolic (SBP), diastolic (DBP), mean arterial (AP) blood pressure and pulse pressure (PP) were calculated. BP in the year of diagnosis was compared to the sum of all BP in subsequent years. In addition, each yearly measurement through the course of AD was compared to its counterpart in the year of diagnosis. Three BP measurements were significantly decreased by from 6.9% to 15.9% in all patients when BP in the year of diagnosis was compared to the sum of each pressure in subsequent years. Sustained BP declines started in the third to fourth year after diagnosis of AD and continued for up to 9 years. The PP was decreased by 19.9% in one patient. There was a strong correlation between the number of C-1 neurons in these cases and their AP and SBP in the years after diagnosis. Hypothalamic phenylethanolamine N-methyltransferase activity was decreased by 63% in AD compared to control cases. Neurofibrillary tangles were found in the paraventricular nucleus of the hypothalamus in an AD case. We postulate that BP is altered in AD as neurons which regulate it degenerate.

Thyroid hormone coordinately regulates Na+-K+-ATPase alpha- and beta-subunit mRNA levels in kidney.

Synthesis of the sodium pump, Na+-K+-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T3) regulates the concentration of the mRNAs coding for the two enzyme subunits, alpha and beta, and the time course of the response. A single dose of T3 (250 micrograms/100 g body wt) was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T3-injected animals, as well as hypothyroid and euthyroid rats, alpha- and beta-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; alpha-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na+-K+-ATPase activity was measured enzymatically. alpha- and beta-mRNAs increased coordinately, after a 6-h time lag to 1.6-fold over hypothyroid levels by 12 h after T3. alpha-Subunit abundance increased significantly by 48 h and to 1.4-fold over hypothyroid by 72 h after T3. Na+-K+-ATPase activity increased with the same time course as the increase in alpha-subunit abundance to 1.3-fold over hypothyroid by 72 h after T3. We conclude that T3 regulates Na+-K+-ATPase synthesis and activity by coordinately increasing the mRNAs of both the alpha- and beta-subunits of the enzyme.