Detection of SARS-CoV-2 IgG antibodies and inflammatory cytokines in saliva-a pilot study.

Objective: The pandemic caused by SARS-CoV-2 virus continues to have a profound effect worldwide. However, COVID-19 induced oral facial manifestations have not been fully described. We conducted a prospective study to demonstrate feasibility of anti-SARS-CoV-2 IgG and inflammatory cytokine detection in saliva. Our primary objective was to determine whether COVID-19 PCR positive patients with xerostomia or loss of taste had altered serum or saliva cytokine levels compared to COVID-19 PCR positive patients without those oral symptoms. Our secondary objective was to determine the correlation between serum and saliva COVID-19 antibody levels. Materials and methods: For cytokine analysis, saliva and serum were obtained from 17 participants with PCR-confirmed COVID-19 infection at three sequential time points, yielding 48 saliva samples and 19 paired saliva-serum samples from 14 of the 17 patients. For COVID-19 antibody analyses, an additional 27 paired saliva-serum samples from 22 patients were purchased. Results: The saliva antibody assay had 88.64% sensitivity [95% Confidence Interval (CI) 75.44%, 96.21%] to detect SARS-CoV-2 IgG antibodies compared to serum antibody. Among the inflammatory cytokines assessed - IL-6, TNF-α, IFN-γ, IL-10, IL-12p70, IL-1ß, IL-8, IL-13, IL-2, IL-5, IL-7 and IL-17A, xerostomia correlated with lower levels of saliva IL-2 and TNF-α, and elevated levels of serum IL-12p70 and IL-10 (p < 0.05). Loss of taste was observed in patients with elevated serum IL-8 (p < 0.05). Conclusions: Further studies are needed to construct a robust saliva-based COVID-19 assay to assess antibody and inflammatory cytokine response, which has potential utility as a non-invasive monitoring modality during COVID-19 convalescence.

Genetic analysis of p53 nuclear importation.

A key step in activation of the p53 tumor suppressor is its transport into the nucleus; however, despite intensive study of p53, the regulation of its subcellular localization is still poorly understood. Here we examined the p53 nuclear importation using a series of mutant cell lines that were resistant to the growth inhibitory effects of temperature-sensitive murine p53 (tsp53). Examination of the p53 subcellular localization in these cell lines showed that the protein was cytoplasmic in most of them. Using a digitonin-permeabilized cell in vitro nuclear import system, we show that cytosols from these cell lines do not support nuclear translocation of a p53 nuclear localization signal (NLS)-containing substrate protein, but promote nuclear localization of a SV40TAgNLS-containing substrate. Complementation assays and use of the mutant cells themselves in the in vitro assays demonstrate that both soluble and insoluble protein components are involved in p53 nuclear import. Collectively, our results suggest that there is a p53 NLS-selective nuclear import pathway and that both soluble and insoluble proteins are involved in its function.

Treatment of AA amyloidosis in rheumatoid arthritis.

Four patients of rheumatoid arthritis (RA) with biopsy confirmed AA amyloidosis were treated with chlorambucil. All had established but uncontrolled RA with a persistently raised ESR. Moderate (> 1 gm, < 3.5 gm/d) to nephrotic range (> 3.5 gm/d) proteinuria and a relatively well preserved renal function was noted in three patients. One patient had deranged renal function and required dialysis. On chlorambucil, there was complete recovery, partial improvement and no improvement in one patient each. The fourth patient required haemodialysis, did not tolerate chlorambucil and succumbed to the illness. Therapy with chlorambucil can benefit some patients of RA with AA amyloidosis. Leucopenia is the most important dose limiting side effect.

Efficacy of isoniazid prophylaxis in patients with systemic lupus erythematosus receiving long term steroid treatment.

OBJECTIVE: To study the efficacy of isoniazid prophylaxis (INHP) in patients with systemic lupus erythematosus (SLE) receiving long term glucocorticosteroid treatment. PATIENTS AND METHODS: Treatment with INHP (5 mg/kg/day, max 300 mg/day) together with pyridoxine 10 mg/day for one year was started in all patients with SLE seen between January 1994 and December 1999 and followed up thereafter. Clinical examination and chest radiography were carried out in all patients before the start of INHP treatment. A liver profile was obtained only if liver toxicity was suspected owing to nausea, loss of appetite, and icterus. Only the data of those patients who completed the INHP treatment or who were withdrawn owing to toxicity have been analysed. This was compared with the results of an earlier study of the incidence of tuberculosis (TB) in patients with SLE not receiving INHP. RESULTS: Ninety seven patients were included, of whom 95 completed one year's treatment with INHP. Treatment was discontinued in two owing to toxicity: hepatitis in one and peripheral neuropathy in one, at eight and 10 months, respectively. One patient developed TB within one month of starting INHP. Seventy patients were followed up further for at least one year (mean 26.4 months, range 12-60 months) after completion of the INHP treatment. During this period one patient developed TB after one month. No deaths due to TB or hepatitis occurred. In comparison with earlier series the incidence of TB decreased from 11% to 2%, a reduction of 82%. The cost of treatment for each case of TB prevented in the first year was 5800 rupees. CONCLUSION: INHP is safe and effective in SLE.

Deoxycholic acid suppresses p53 by stimulating proteasome-mediated p53 protein degradation.

Bile acids, principally deoxycholic acid (DCA), have been implicated in the promotion of colon tumorigenesis in both animals and humans. Increasing evidence suggests that bile acids may exert their tumor promoting activity by modulating intracellular signaling and altering gene expression. In this study we have investigated the effect of bile acids on the tumor suppressor p53 using the human colon tumor cell line HCT116, which retains the wild-type p53 gene and functional p53 signaling in response to DNA damage. We found that exposure of the cells to elevated concentrations of DCA suppressed accumulation of p53 protein as well as p53 transactivation and impaired the p53 response of the cells to DNA damaging agents, such as ionizing radiation. Neither ursodeoxycholic acid, a putative chemopreventive agent, nor cholic acid, which is biologically inert, had any effect on p53 protein level and transactivation activity. Further examination revealed that instead of inhibition, DCA induced p53 mRNA in a dose-dependent manner, indicating that the inhibitory effect of DCA on p53 protein is mediated by a post-transcriptional mechanism. Both lactacystin, a specific inhibitor of the 26S proteasome, and leptomycin B, a specific inhibitor of the nuclear export protein CRM1, could block the effect that DCA had on p53 protein levels, suggesting that DCA suppressed p53 by stimulating the process of proteasome-mediated degradation of p53. Significantly, blocking extracellular signal-regulated kinase (ERK) signaling, but not protein kinase C (PKC), blunted suppression by DCA of p53 protein levels and transactivation activity, suggesting that DCA suppressed p53, in part, by stimulating the ERK signaling pathway. Both ERK and PKC signaling have been previously demonstrated to be stimulated by DCA. These results suggest a novel signaling mechanism of bile acids that may play an important role in colon tumor promotion mediated by bile acids.

Morphologic conversion of a neuroblastoma-derived cell line by E6-mediated p53 degradation.

Neuroblastoma-derived tumor cells, unlike cells from other tumor types, characteristically express a wildtype but cytoplasmically sequestered p53 protein. To ascertain whether the p53 in these cells retained any physiological activity, we inactivated it in SK-N-SH cells, a neuroblastoma-derived cell line, by introducing the human papilloma virus type 16 E6 expression plasmid. Parent SK-N-SH cell cultures are composed of two cell types exhibiting characteristic morphologies designated neuroblastic (N-type) or substrate-adherent fibroblastic (S-type) cells, both of which have been shown to spontaneously transdifferentiate or interconvert. We report here that down-regulation of p53 resulted in conversion of SK-N-SH cells to the substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, and a lack of responsiveness to retinoic acid-induced neuronal differentiation. Importantly, we did not observe N-type cells in the E6-transfected cell population, suggesting that they were incapable of transdifferentiating to the N-type morphology. We also tested the ability of these E6-transfected S-type cells to form colonies in soft agar and observed a markedly reduced capacity of these cells to do so when compared with the parent and mutant E6-transfected cells. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype.

Adrenal histoplasmosis.

A 60 year old diabetic was admitted with the history of low grade fever and weight loss of six weeks duration. After investigations, he was detected to have bilateral adrenal masses which on biopsy proved to be due to histoplasmosis. He was treated with itraconazole and made complete recovery.

Conformational phenotype of p53 is linked to nuclear translocation.

P53 is inactivated in tumors by mechanisms other than mutations in the p53 gene itself. To gain insight into the mechanisms by which this inactivation occurs, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. We expanded 22 clones (ALTR cells for A1-5 Low Temperature Resistant) that could grow at the permissive temperature. Most exhibited cytoplasmic sequestration as the mechanism by which p53 was inactivated. We show here that this cytoplasmically sequestered tsp53 protein is maintained in a mutant conformation. Only in clones with nuclear localized p53 is it also expressed in the wild-type conformation suggesting that subcellular localization of tsp53 is important in determining the conformation of the protein. Consistent with this, we show that the changes in conformation of p53 in A1-5 and SK-N-SH cells induced by ionizing radiation also correlate with nuclear translocation of p53. We suggest that nuclear translocation of p53 can result in a change in the conformation from mutant to wild-type but that these may be two separable events. Oncogene (2000) 19, 4042 - 4049.

Isolation of bacteria other than Helicobacter pylori from stomachs of squirrel monkeys (Saimiri spp.) with gastritis.

Gastric biopsy specimens obtained from 12 squirrel monkeys (Saimiri spp.) were investigated by culture for the presence of bacteria. The stomachs of two monkeys with gastritis were colonized with gram-negative, urease-positive bacteria, identified as Ochrobactrum anthropi by the Vitek and API NFT methods (BioMérieux). A third monkey with gastritis was positive for Aeromonas salmonicida and Pseudomonas vesicularis (both urease-negative). No Helicobacter pylori was isolated from squirrel monkeys. Light microscopic and transmission electron microscopic examination revealed that the O. anthropi isolates were covered by extracellular material, indicating a capsule. Characterization of the O. anthropi urease revealed Michaelis-Menten constants (Km values) of 6.2 and 4.0 mM urea for the ureases of O. anthropi isolates S664 and S1835, respectively, and 3.7 for type strain 49188. Western blot analysis using H. pylori- and H. felis-specific antibodies detected shared antigenic epitopes between the ureases of H. pylori, H. felis, and O. anthropi. The apparent molecular mass of the urease enzymes of the O. anthropi isolates was determined on 6% nondenaturing gels to be approximately 82 kDa. Antimicrobial susceptibility tests, using the MicroScan method (Dade International), revealed multidrug resistance for the O. anthropi isolates with susceptibilities for the antibiotics amikacin, ciprofloxacin, gentamicin, cefoperazone, tobramycin, imipenem, and trimethoprim/sulfamethoxazole.