Novel germline variants of CDKN1B and CDKN2C identified during screening for familial primary hyperparathyroidism.

PURPOSE: CDKN1B mutations were established as a cause of multiple endocrine neoplasia 4 (MEN4) syndrome in patients with MEN1 phenotype without a mutation in the MEN1 gene. In addition, variants in other cyclin-dependent kinase inhibitors (CDKIs) were found in some MEN1-like cases without the MEN1 mutation. We aimed to describe novel germline mutations of these genes in patients with primary hyperparathyroidism (PHPT). METHODS: During genetic screening for familial hyperparathyroidism, three novel CDKIs germline mutations in three unrelated cases between January 2019 and November 2021 were identified. In this report, we describe clinical features, DNA sequence analysis, and familial segregation studies based on these patients and their relatives. Genome-wide DNA study of loss of heterozygosity (LOH), copy number variation (CNV), and p27/kip immunohistochemistry was performed on tumour samples. RESULTS: DNA screening was performed for atypical parathyroid adenomas in cases 1 and 2 and for cystic parathyroid adenoma and young age at diagnosis of PHPT in case 3. Genetic analysis identified likely pathogenic variants of CDKN1B in cases 1 and 2 and a variant of the uncertain significance of CDKN2C, with uniparental disomy in the tumour sample, in case 3. Neoplasm screening of probands showed other non-endocrine tumours in case 1 (colon adenoma with dysplasia and atypical lipomas) and case 2 (aberrant T-cell population) and a non-functional pituitary adenoma in case 3. CONCLUSION: Germline mutations in CDKIs should be included in gene panels for genetic testing of primary hyperparathyroidism. New germline variants here described can be added to the current knowledge.

Low frequency of positive antithyroid antibodies is observed in patients with thyroid dysfunction related to immune check point inhibitors.

INTRODUCTION: Immune checkpoint inhibitors (ICI), such as programmed death-1 inhibitors (anti-PD1), have become a cornerstone for the treatment of different advanced cancers. These antibodies act as modulators of immune checkpoint proteins. However, ICI can lead to the breaking of immune self-tolerance, inducing autoimmune side effects (irAEs), including endocrinopathies. One of the most frequent endocrine irAE of anti-PD1 is thyroid dysfunction, but the exact mechanism of this disease still remains unknown. MATERIALS AND METHODS: We conducted a descriptive retrospective study, analyzing 11 patients who received at least one dose of anti-PD1 (nivolumab or pembrolizumab) and presented thyroid irAEs. Data were collected between September 2015 and May 2018 in our hospital. The aim was to analyze the clinically relevant features of thyroid irAEs and the frequency of antithyroid antibodies (ATA) positivity observed on them. RESULTS AND DISCUSSION: 8 of the 11 patients were treated with nivolumab and the other three patients received pembrolizumab. Six patients presented silent thyroiditis with a thyrotoxicosis phase; three patients developed directly primary/subclinical hypothyroidism and two patients showed primary hyperthyroidism. Thyroid autoantibodies (anti-Thyroglobulin and anti-Thyroid Peroxidase) were assessed in all the 11 patients, and only in two of them (18%) a positive titer was displayed. Anti-TSH receptor antibodies (TRAbs) were examined in five patients, three with painless thyroiditis at the time of thyrotoxicosis and two with primary hyperthyroidism, and they all had undetectable levels. CONCLUSIONS: In our sample of 11 Caucasian patients with thyroid dysfunction related with anti-PD1, we found low frequency of ATA positive titers, comparable to other recent reports in others ethnicities, which could suggest that silent thyroiditis due to pembrolizumab or nivolumab has a different pathogenesis from the classical autoimmune spontaneous thyroiditis. Further investigations are required to completely understand the immune mechanisms involved.

The assessment of zinc status by the zinc tolerance test in various groups of patients.

The zinc status of young and aged subjects, hypertensives, geriatric patients with leg ulcers and cancer patients has been determined by various means. Serum, saliva, urine and hair zinc were measured by atomic absorption spectrometry and no correlation was observed between the zinc levels in any of these differing matrices. The mean hair and salivary zinc level showed little variation between the differing groups of patients and provided little or no indication of zinc status. The results of the present experiment indicate that the zinc tolerance test, that is, an unequivocal rise in serum zinc following per oral administration of this metal, provides the best indication of zinc status.

Vitamin E retards the lipoperoxidation resulting from anticancer drug administration.

The administration of either 5-fluorouracil, methotrexate, cyclophosphamide or vincristine to rats produced an increase in liver and plasma, but not brain, lipoperoxide levels. There was no significant difference between the glutathione peroxidase activity in the liver and the brain tissue of cytotoxic drug-treated and control rats. Glutathione peroxidase activity was significantly lower in the erythrocytes of 5-fluorouracil-and methotrexate-treated rats than in control animals. The erythrocyte glutathione peroxidase levels of vincristine- and cisplatin-treated rats did not differ significantly from the control levels. Rats which received vitamin E supplementation concomitantly with 5-fluorouracil treatment had liver and plasma lipoperoxide levels which were significantly lower than those which had received only the anticancer drug. The tissue lipoperoxide levels in the vitamin E supplemented, 5-fluorouracil-treated rats were comparable with those of arachis oil-treated controls.

The influence of zinc status on the anti-Lewis lung tumour activity of cisplatin and gallium.

Treatment of C57/BL mice with either cisplatin or gallium nitrate inhibited the growth and metastasis of the Lewis lung tumour and these anti-tumour agents also lowered the zinc levels of some tissues. Nutritional zinc deficiency or the deficiency arising from treatment with the chelating agent, penicillamine, also restricted tumour growth. Although the anti-tumour activity of cisplatin was enhanced in zinc-deficient mice, many of these animals died before sacrifice 14 days after tumour inoculation. The results indicate that zinc status could have considerable bearing on the therapeutic index of the mental-containing anti-cancer drugs.

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering.

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants were used. Transient expression of ß-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering with this crop.

The influence of electrostimulation on hexobarbital induced loss of righting reflex in rats.

Electrostimulation (ES) of slow (SF, 10 Hz) or fast (FF, 500 Hz) frequency decreases the sleeping time of rats anaesthetized by administration of acute doses of hexobarbital. When ES is applied via the ears, both SF and FF are equally efficient in reducing the loss of righting reflex (LRR), whereas if ES is applied peripherally via the paws, only FF decreases the acute narcosis time. Applied cranially, either continuous stimulation or administration of intermittent current (5 minutes on and off) were equally effective in reducing narcosis. A decreased period of 30 minutes' continuous stimulation will reduce sleeping time only if administered immediately after LRR. When restrained animals received ES for periods of up to 3 hours prior to administration of the barbiturate, the sleeping time of the stimulated and sham treated animals were not significantly different.

Sex differences in the glutathione peroxidase activity of various tissues of the rat.

The glutathione peroxidase activity of blood, liver and brain of sexually mature male and female rats has been determined using both hydrogen peroxide and cumene hydroperoxide as substrates. No significant differences were found on comparison of the glutathione peroxidase activity of the brain tissues of male and female animals. Observed mathematically significant differences between the enzyme activity in the blood of male and females were probably of little physiological significance. Both selenium and non-selenium dependent glutathione peroxidase activities were significantly higher in the livers of female rats but this was much more apparent when cumene hydroperoxide was reduced. Castrated male rats had significantly higher hepatic enzyme activities, approaching those of the female animals. The lipoperoxide concentration was significantly higher in the livers of female rats.

Lipoperoxide levels, glutathione status and glutathione peroxidase activity in liver and tumors of mice bearing the Lewis lung carcinoma.

Lipoperoxides, glutathione status and glutathione peroxidase activity have been determined in normal and neoplastic tissues of control and tumor-bearing mice, tissues from both groups being assayed 5, 7, 9, 11, 13 and 15 days after inoculation. The ratio of hepatic reduced: oxidized glutathione increased in tumor-bearing animals as the tumor increased in size. This ratio was 2.5-fold higher at 15 days than at 10 days after tumor inoculation. In both tumor and hepatic tissue the alteration in the ratio was the result of both an increase in reduced glutathione and a decrease in oxidized glutathione levels. In tumor tissue the progressively increasing reduced glutathione content correlated closely with tumor growth. The presence of a tumor did not significantly affect hepatic glutathione peroxidase activity and there was no significant difference between tumor enzyme activity assayed at 2-day intervals between 9 and 15 days after inoculation. The livers of tumor-bearing animals had significantly higher lipoperoxides than control mice, the levels increasing progressively with tumor growth. Tumor lipoperoxides were also high, usually in excess of the hepatic level. The lungs of nontumored littermates, which were compared with the carcinoma as reference tissue, showed no significant change in either glutathione peroxidase activity or lipoperoxide levels when monitored over the same period.

Abnormal antioxidant defence in some tissues of congenitally obese mice.

The concentration of lipoperoxides (estimated as thiobarbituric acid-reactive material) and some components of the antioxidant defence system have been compared in various tissues of lean and congenitally obese mice. NADPH-stimulated lipoperoxide generation in vitro was significantly higher in microsomes (microsomal fractions) prepared from obese hepatic tissue than lean. Plasma, liver and brain lipoperoxide concentration was significantly higher in obese mice. In blood derived from obese mice the concentration of non-enzymic antioxidants including caeruloplasmin and vitamin A was higher, but hepatic retinol concentration was lower in these animals. In all the tissues assayed the glutathione peroxidase activity against H2O2 was less than its activity against cumene hydroperoxide. Assayed with either substrate, glutathione peroxidase activity was significantly higher in the brain and blood of obese mice than their lean counterparts. Conversely, liver glutathione peroxidase was decreased in obese animals, representing 43% of the activity of the lean-mouse liver enzyme against H2O2 and 81% of the cumene hydroperoxide-reducing activity. The liver of obese mice had significantly less, and the kidneys more, oxidized glutathione than the corresponding tissues of lean mice. Further investigations on hepatic tissue indicated that glutathione reductase activity was lower in the obese animals, but there was no significant difference between glucose-6-phosphate dehydrogenase activity in obese and lean mice.

Superoxide dismutase activity, caeruloplasmin activity and lipoperoxide levels in tumour and host tissues of mice bearing the Lewis lung carcinoma.

Superoxide dismutase (SOD) activity, plasma caeruloplasmin activity and the level of whole tissue and subcellular lipoperoxides have been determined in normal and neoplastic tissues from control and tumour-bearing mice, measurements being made nine, twelve and fifteen days after the inoculation of Lewis lung carcinoma cells. SOD activity of host liver and lung tissues did not vary significantly from those of the control animals. Blood SOD activity of the tumoured animals was markedly elevated on the ninth and twelfth days after inoculation, decreasing to control levels on the fifteenth day. Tumor SOD diminished from activity on the ninth day which was greater than that for control lung to a level significantly lower than that for control lung on the twelfth and fifteenth days after inoculation. The presence of a tumor did not appear to affect plasma caeruloplasmin oxidase levels. The lipoperoxide level of hepatic tissue rose significantly as the tumour progressed. In the lung tissue the lipoperoxides decreased from a level four times higher on the ninth day to one not significantly different from that of the controls. Tumour lipoperoxides were about twice the level of hepatic tissue and of the order of ten-fold greater than those of lung. The level of lipoperoxide in the plasma of tumoured mice did not differ markedly from that of control mice. Assays of lipoperoxide in subcellular fractions of liver, lung and tumour tissue revealed that the elevated lipoperoxide was principally synthesized in the endoplasmic reticulum.

The influence of cold restraint stress on some components of the antioxidant defence system in the tissues of rats of various ages.

Rats of various ages were subjected to stress by confinement in restraining cages at 2-4 degrees C. Analysis of the plasma of these animals revealed an elevation in corticosteroids of approximately 50% above the control level. The livers of all the groups of cold-restrained animals contained significantly more lipoperoxide (estimated as thiobarbituric-acid-reactive material) than did control hepatic tissue. The plasma of the 12-, 24-, and 32-wk-old groups of rats subjected to stressful treatment also contained significantly higher lipoperoxide levels. There was no significant difference between the lipoperoxide levels of the brain tissue of control or stress-treated rats. The activities of both glutathione peroxidase and glutathione reductase were increased in hepatic, but not brain, tissue of the stressed animals. The perturbation of the activities of these enzymes did not produce any significant change in the ratio of reduced, oxidized glutathione. The livers of the stressed animals had significantly less total glutathione than those of the controls.

The amelioration of the suffering associated with spinal cord injury with subperception transcranial electrical stimulation.

STUDY DESIGN: Double blind, partial crossover. OBJECTIVES: To evaluate the analgesic activity of a novel cranial electrostimulus in people with spinal cord injury (SCI). SETTING: Hereward College, a residential centre that provides educational facilities for students with disabilities. METHODS: Subjects with SCI experiencing chronic pain were randomly assigned into two groups, one of which received sham and the other transcranial electrostimulation treatment (TCET) on two occasions daily for four successive days. After a 'wash-out' period of 8 weeks all subjects returned and received the identical stimulus that the treated cohort received on the first arm of the study. RESULTS: Pain measurements applied before and after each session indicated that the pain decreased in the treated group to 51% of that reported at the commencement of treatment; reported pain intensity did not decrease significantly in the sham treated subjects. The same (sham) subject group reported experiencing 59% of the pain at the end of the second arm of the study (TCET) as on the first arm (sham). No significant differences were determined between the mood of all subjects estimated before and after each sham or TCET treatment session. The reported analgesic, and combined antidepressant and anxiolytic drug use in subjects receiving TCET on the second arm of the study, was 46% and 53% respectively of the average pre-study drug use. No similar decrease in the use of the drugs was noted in the same subjects after sham treatment on the first arm of the study. Salivary cortisol determinations made prior to and after each sham and treatment session implicated this corticoid in the pain-relieving mode of action of the treatment, but could not be associated with any changes in mood. Subjects receiving TCET had significantly higher urinary 3-methoxy-4-hydroxy-phenylglycol (MHPG) output after the TCET treatment period than sham stimulation, implicating increased central noradrenaline (NA) metabolism in the observed effects. CONCLUSION: The subjects reported less pain during, and immediately after receiving this transcranial treatment, although they were using less medication than when receiving sham treatment.

The effect of isolation stress on some hepatic drug and carcinogen metabolising enzymes in rats.

Rats were subjected to stress by isolation for periods of up to eight days, which produced an elevation in plasma cortisol. In vivo drug metabolism as estimated by the plasma elimination rate of orally-administered antipyrine was not significantly affected by this treatment although there was an apparent decrease in the absorption rate of the drug. In vitro experiments on hepatic microsomal preparations derived from stressed animals indicate that this stress increased in the activity of some enzyme systems concerned with benzo(a)pyrene activation and this correlated with an increased binding of the carcinogen to DNA. The activity of conjugating enzyme which could catalyze the excretion of such carcinogens was not significantly altered. The results indicated that stress could have an important bearing on carcinogenesis by enhancing to a greater extent enzyme systems responsible for activation than those involved in the excretion of polycyclic aromatic hydrocarbons.

The effect of chloroform inhalation on hepatic glucuronidation and de-glucuronidation mechanisms.

The effect of 2, 4, 6 or 8 exposures to chloroform vapour on hepatic glucuronidating (UDPGA transferase) and de-glucuronidating (beta-glucuronidase) levels has been studied in rats. Successive treatments progressively decreased hepatic UDPGA transferase to a minimum of 53% of the control level. beta-Glucuronidase activity was increased two-fold after only two exposures and remained elevated for subsequent exposures. Cytochrome P450 levels decreased with each exposure. The level of this coenzyme in the treated animals remained lower than that of the control animals for at least 48 hours after treatment. UDPGA transferase was diminished to its lowest levels 9 hours after the final exposure to chloroform and did not achieve the control value for a further 48 hours. The beta-glucuronidase activity remained elevated for 12 hours after final exposure. The present experiment demonstrates that inhalation of toxic solvents such as chloroform decreases the glucuronidating capacity of the liver.

Hepatic function assessed (in rats) during chemotherapy with some anti-cancer drugs.

Using rats, we studied how best to assess hepatic damage after administering therapeutic doses of each of five anti-cancer drugs or of the hepatotoxin, carbon tetrachloride. As indexes, we compared measurement of the concentration of administered antipyrine in plasma with measurement in serum of alpha-fetoprotein or of the activities of five enzymes that reportedly best reflect hepatic damage. The biological half-life of antipyrine in the plasma was increased more than threefold on pretreating the rats with any of the five cytotoxic drugs or with carbon tetrachloride. In contrast, the concentrations of alpha-fetoprotein, alkaline phosphatase, gamma-glutamyltransferase, or glutamate dehydrogenase were not consistently increased. Of the enzymes tested in serum, aspartate aminotransferase and ornithine carbamoyltransferase best indicated hepatic impairment resulting from the treatment with anti-cancer drugs. Our results imply that determination of the pharmacokinetics of marker drugs such as antipyrine better indicates hepatic dysfunction induced by cytotoxic agents than does measurement of the enzymes liberated into serum as a result of damage to liver mitochondria.

The effect of chronic alcohol intake upon the hepatic microsomal carcinogen-activation system.

Ethanol was administered to mice either by repeated intraperitoneal injection, or orally in the drinking water over an extended period of time. Following intraperitoneal ethanol pre-treatment further groups of mice received an injection of benzo(a)pyrene. Alcohol intake decreased the level of microsomal aryl hydrocarbon hydroxylase which corresponded to the observed decrease in DNA binding of benzo(a)pyrene. In contrast, the number of tumors which developed in the alcohol pre-treated mice exceeded those of the control animals.