Survivin expression in odontogenic keratocysts and correlation with cytomegalovirus infection.

OBJECTIVES: The aim of this study was to investigate the expression of survivin, an inhibitor of apoptosis, in odontogenic keratocysts and to compare it to the findings in non-neoplastic jaw cysts - periapical cysts, as well as to establish a possible relationship between survivin expression and human cytomegalovirus presence within these cysts. MATERIALS AND METHODS: Samples of 10 odontogenic keratocysts (five positive and five negative for the presence of cytomegalovirus, as determined by polymerase chain reaction) and 10 periapical cysts (five positive and five negative for the cytomegalovirus presence) were analysed. The expression of survivin was assessed by immunohistochemical methods, using monoclonal antibody that selectively recognizes the cytoplasmic form of survivin. RESULTS: All 10 odontogenic keratocysts showed immunostaining for survivin, while all 10 periapical cysts were negative for its presence. There was no correlation between cytomegalovirus presence and expression of survivin within odontogenic keratocysts. CONCLUSION: Survivin may contribute to the aggressive behavior of odontogenic keratocysts, and thus support the emerging opinion of their neoplastic nature.

Autonomic balance determines the severity of COVID-19 courses.

COVID-19 has left mankind desperately seeking how to manage dramatically rising infection rates associated with severe disease progressions. COVID-19 courses range from mild symptoms up to multiple organ failure and death, triggered by excessively high serum cytokine levels (IL 1ß, IL 6, TNF α, IL 8). The vagally driven cholinergic anti-inflammatory pathway (CAP) stops the action of nuclear factor κB (NF-κB), the transcriptional factor of pro-inflammatory cytokines. Thus, well-balanced cytokine release depends on adequate vagal signaling. Coronaviruses replicate using NF-κB transcriptional factor as well. By degrading the cytoplasmatic inhibitor of NF-κB subunits (IκB), coronaviruses induce unrestricted NF-κB expression accelerating both, virus replication and cytokine transcription.We hypothesize that CAP detriment due to depressed vagal tone critically determines the severity of COVID-19.

Long-term outcome in Serbian patients with Wilson disease.

BACKGROUND AND PURPOSE: To investigate survival rates, prognostic factors, and causes of death in Wilson disease (WD). METHODS: In the years 1980-2007, a cohort of 142 patients with WD was prospectively registered (54 presented with neurologic symptoms, 49 with hepatic symptoms, 33 had mixed form, and data were missing for six patients). The duration of follow-up for patients alive was 11.1 +/- 8.8 years. RESULTS: After initiation of treatment (d-penicillamine and zinc salts), 79% of patients had a stable or improved course of disease. Despite early diagnosis and appropriate therapy, 15 patients still had a relentlessly progressive course. Thirty patients died. The cumulative probability of survival in a 15-year period for the whole group was 76.7 +/- 4.9%. Better prognosis of WD was associated with male sex, younger age at onset, neurologic form of the disease, and treatment continuity. Causes of death were predominantly related to hepatic failure (16 patients), but also suicide (four patients) and cancer (three patients). CONCLUSION: Despite the relatively early diagnosis and treatment of our patients with WD, mortality was still considerably high.

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

Buffered Romanowsky-Giemsa method for formalin fixed, paraffin embedded sections: taming a traditional stain.

Romanowsky-Giemsa (RG) stains were devised during the 19th century for identifying plasmodia parasites in blood smears. Later, RG stains became standard procedures for hematology and cytology. Numerous attempts have been made to apply RG staining to formalin-fixed paraffin-embedded tissue sections, with varied success. Most published work on this topic described RG staining methods in which sections were overstained, then subjected to acid differentiation; unfortunately, the differentiation step often caused inconsistent staining outcomes. If staining is performed under optimal conditions with control of dye concentration, pH, solution temperature and staining time, no differentiation is required. We used RG and 0.002 M buffer, pH 42, for staining and washing sections. All steps were performed at room temperature. After staining and air drying, sections were washed in 96-100% ethanol to remove extraneous stain. Finally, sections were washed in xylene and mounted using DPX. Staining results were similar to routine hemalum and eosin (H & E) staining. Nuclei were blue; intensity depended largely on chromatin density. RNA-rich sites were purple. Collagen fibers, keratin, muscle cells, erythrocytes and white matter of the central nervous system were stained pinkish and reddish hues. Cartilage matrix, mast cell granules and areas of myxomatous degeneration were purple. Sulfate-rich mucins were stained pale blue, while those lacking sulfate groups were unstained. Deposits of hemosiderin, lipofuscin and melanin were greenish, and calcium deposits were blue. Helicobacter pylori bacteria were violet to purple. The advantages of the method are its close similarity to H & E staining and technical simplicity. Hemosiderin, H. pylori, mast cell granules, melanin and specific granules of different hematopoietic cells, which are invisible or barely distinguishable by H & E staining, are visualized. Other advantages over previous RG stains include shorter staining time and avoidance of acetone.

The role of multiply phosphorylated S6 in ribosome degradation.

Rat liver ribosomes, prepared 1-24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure.

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.

Novel DNA-binding protein from Drosophila embryos identified by binding site selection.

The DNA sequence C/AGAGCGC/AGA, related to binding sites for GAF and Zeste transcription factors, was selected from a pool of degenerate PCR fragments for binding to the cytoplasmic protein of Drosophila preblastoderm embryos. Identical DNA binding activity was also detected in embryonic nuclei. Based on several criteria, such as size, intracellular distribution, sensitivity to ATP and protein kinase inhibitor 6-DMAP, kinetics during development and lack of cross-reaction with rabbit anti-GAF serum, protein recognizing selected sequence was shown to differ from either Zeste or GAF.

Disease modifying and immunomodulatory effects of high dose 1 alpha (OH) D3 in rheumatoid arthritis patients.

OBJECTIVE: Vitamin D analogues such as 1 alpha (OH) D3 (alphacalcidiol) have a possible physiological paracrine effect on cell proliferation and differentiation. Experimentally established possibilities to prevent autoimmune diseases suggest that alphacalcidiol may have therapeutic value as an immunomodulatory agent in patients with rheumatoid arthritis. METHODS: We organized a 3-month open-label trial on 19 patients being treated with standard DMARD therapy for acute RA. They were divided into 2 subgroups, those with highly active RA and those with moderately active RA. Their regular drug regimen was maintained during the trial and oral alphacalcidiol 2 micrograms/day was added. Therapy results were evaluated by ESR, CRP, morning stiffness, the Richie index, and the Lee index. Immunomodulatory effects were investigated by measuring lymphocyte proliferation and apoptosis both in the patients and in vitro in 10 nM alphacalcidiol-supplemented culture medium. RESULTS: After 3 months, high dose oral alphacalcidiol therapy showed a positive effect on disease activity in 89% of the patients (45% or 9 pts. with complete remission and 44% or 8 pts. with a satisfactory effect). Only two patients (11%) showed no improvement, but no new symptoms occurred. No side effects were observed. CONCLUSION: These results suggest that alphacalcidiol is a powerful immunomodulatory agent with fairly low hypercalcemic activity. Clinical improvement was strongly correlated with the immunomodulating potential of this agent. We noticed dual effects on lymphocyte proliferation and apoptosis according to the prior cell activation state. Alphacalcidiol could therefore possibly be used as an adjunct therapy with DMARDs in patients with rheumatoid arthritis.

The pharmacologic stability of 35-year old theophylline.

The propensity to preserve and to hoard drugs over the years at home is a well-known phenomenon and offers the possibility for intentional and accidental drug poisoning in man. We report a case of acute theophylline poisoning in an 80-year old women after ingestion of 'Asthmo-Kranit', a 35-year old combined preparation containing theophylline and aminopyrine as the main ingredients. The patient developed the typical clinical picture of a symptomatic theophylline poisoning with flush, tremor, tachycardia, hyperventilation, hypotonia, and hyperglycaemia. The clinical course after treatment with beta-blockers was without complications. The determination of theophylline in tablets showed stability of 90% of the labelled amount of the drug 30 years beyond the expiration date. The case illustrates the prolonged shelf stability and pharmacological potency of some pharmaceuticals and points to the risk of long-outdated prescriptions. Physicians should primarily not underestimate drug toxicity in consequence of old-age pharmaceuticals.

[Conjunctivitis lignosa and hydrocephalus (an unusual association of 2 diseases)]. / Conjunctivitis lignosa i gidrotsefaliia (neobychnaia assotsiatsiia dvukh boleznei).

Two siblings, a brother and a sister, with ligneous conjunctivitis and hydrocephalus coursing in parallel were described. Both conditions developed during the first year of life. Surgery for hydrocephalus was carried out in both children and conjunctivitis gradually subsided.

Acute toxic effects of single dose dacarbazine: hematological and histological changes in an animal model.

Treatment of advanced soft tissue sarcoma usually includes dacarbazine (DTIC), an alkylating agent that methylates DNA and is active during all phases of the cell cycle. Common side effects of DTIC include nausea, vomiting, impaired liver and kidney function, myelosuppression, and pneumonia. There are no accounts, however, of histological and hematological changes caused by DTIC. We investigated acute hematological and morphological changes in different organs and in tumors that were caused by a single dose of DTIC. Adult Syrian golden hamsters were inoculated with a suspension of tumorigenic baby hamster kidney (BHK) cells by subcutaneous injection. On day 14 after inoculation, doses of 1.4, 1.6, 1.8 or 2.0 g/m(2) DTIC were injected intraperitoneally into the hamsters. Hamsters in the control group were injected with physiological saline in the same way. Seven days after drug or saline injection the animals were sacrificed and samples of blood, heart, kidney, liver, lungs, spleen, small intestine and tumor were excised, processed and analyzed. Mitoses were counted using an ocular extension with engraved frame. Anemia, thrombocytopenia and leukocytosis were found in the control group of hamsters with fibrosarcoma, whereas animals with fibrosarcoma treated with DTIC developed anemia, thrombocytopenia and leukopenia. Severe pneumonia and moderate hepatitis were detected in all DTIC treated groups. Effects of DTIC on tumor cells included rounding and enlargement of nuclei and rarefaction of chromatin. The number of mitoses was reduced with increasing doses of DTIC. Hepatitis, myelosuppression, pneumonia, and dose-related inhibition of tumor cell proliferation were observed after a single dose of DTIC.

Romanowsky-Giemsa as a counterstain for immunohistochemistry: optimizing a traditional reagent.

We describe a detailed protocol for using Romanowsky-Giemsa (RG) counterstaining on formalin fixed, paraffin embedded tissue sections that are stained immunohistochemically (IHC) after antigen retrieval using hot acidic citrate buffer. RG staining is easy to perform and provides consistent results that are similar to hematoxylin and eosin (HE) staining. The counterstaining was applied after a variety of antibodies that used the DAB chromogen and the intensity of IHC stained structures was preserved. Moreover, RG counterstaining provided finer cell detail than HE, methyl green or nuclear fast red. A detailed troubleshooting guide is provided for the RG staining protocol.