Virtual histological staining of unlabeled autopsy tissue.

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Post-Chemotherapy Histiocyte-Rich Pseudotumors: Radiologic and Endoscopic Mimics of Residual Lymphoma.

Development of post-chemotherapy histiocyte-rich pseudotumor (PHP) is an underrecognized event following therapy in lymphoma patients and may mimic residual tumor using current therapy monitoring protocols. We report a series of 5 patients with PHP along with a review of the literature. In our series, we describe 3 patients with persistent hypermetabolic masses by positron emission tomography-computed tomography, one with persistent terminal ileal nodules on endoscopy, and one with bone marrow involvement, a site not associated with mass-like disease. Twenty-three patients with long-term follow-up were identified from our series and review of the literature. Forty-four percent of patients received additional therapy, and only 4% of patients died of lymphoma. This study illustrates that PHPs are not identified using current lymphoma therapy monitoring algorithms and may result in overtreatment with risk for additional therapy-related complications. The need for confirmatory tissue biopsy in this setting is recommended.

Loss of miR-24-3p promotes epithelial cell apoptosis and impairs the recovery from intestinal inflammation.

While apoptosis plays a significant role in intestinal homeostasis, it can also be pathogenic if overactive during recovery from inflammation. We recently reported that microRNA-24-3p (miR-24-3p) is elevated in the colonic epithelium of ulcerative colitis patients during active inflammation, and that it reduced apoptosis in vitro. However, its function during intestinal restitution following inflammation had not been examined. In this study, we tested the influence of miR-24-3p on mucosal repair by studying recovery from colitis in both novel miR-24-3p knockout and miR-24-3p-inhibited mice. We observed that knockout mice and mice treated with a miR-24-3p inhibitor had significantly worsened recovery based on weight loss, colon length, and double-blinded histological scoring. In vivo and in vitro analysis of miR-24-3p inhibition in colonic epithelial cells revealed that inhibition promotes apoptosis and increases levels of the pro-apoptotic protein BIM. Further experiments determined that silencing of BIM reversed the pro-apoptotic effects of miR-24-3p inhibition. Taken together, these data suggest that miR-24-3p restrains intestinal epithelial cell apoptosis by targeting BIM, and its loss of function is detrimental to epithelial restitution following intestinal inflammation.

Anetoderma in a patient with a history of primary syphilis.

Anetoderma is a rare cutaneous disorder presenting with atrophic skin lesions. It can be associated with several autoimmune and infectious diseases. With the current resurgence of syphilis, clinicians must be aware of its association with anetoderma.

Occult Colonic Perforation in a Patient With Coronavirus Disease 2019 After Interleukin-6 Receptor Antagonist Therapy.

BACKGROUND: Interleukin-6 blockade (IL-6) has become a focus of therapeutic investigation for the coronavirus disease 2019 (COVID-19). METHODS: We report a case of a 34-year-old with COVID-19 pneumonia receiving an IL-6 receptor antagonist (IL-6Ra) who developed spontaneous colonic perforation. This perforation occurred despite a benign abdominal exam and in the absence of other known risk factors associated with colonic perforation. RESULTS: Examination of the colon by electron microscopy revealed numerous intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions abutting the microvilli of the colonic mucosa. Multiplex immunofluorescent staining revealed the presence of the SARS-CoV-2 spike protein on the brush borders of colonic enterocytes that expressed angiotensin-converting enzyme 2. However, no viral particles were observed within the enterocytes to suggest direct viral injury as the cause of colonic perforation. CONCLUSIONS: These data and absence of known risk factors for spontaneous colonic perforation implicate IL-6Ra therapy as the potential mediator of colonic injury in this case. Furthermore, this report provides the first in situ visual evidence of the virus in the colon of a patient presenting with colonic perforation adding to growing evidence that intact infectious virus can be present in the stool.

Neutrophilic figurate erythema of infancy: A diagnostic challenge.

Neutrophilic figurate erythema of infancy (NFEI) is a rare variant of annular erythema of infancy. It is characterized by annular erythematous plaques, occasionally with a polycyclic configuration. The main challenge is to differentiate this rare entity from other figurate erythemas associated with serious diseases such as neonatal lupus erythematosus. We present the case of a 9-month-old female admitted with a skin rash of unclear etiology. The rash started on her face at the age of 3 months and gradually spread to her extremities. She had no constitutional symptoms, and her health and development were otherwise unremarkable since birth. This persistent skin eruption consisted of many ill-defined erythematous papules and annular plaques. Histologic examination revealed perivascular neutrophils and eosinophils with abundant nuclear dust without signs of vasculitis. NFEI is a diagnostic enigma both clinically and histologically. Absence of an underlying cause, dermal neutrophilic infiltrate with leukocytoclasis, and lack of vascular damage are the keys to diagnosis.

Hydrophilic polymer microemboli in a patient with a chronic cutaneous ulcer: a case report.

Hydrophilic polymer coatings were designed to reduce friction between the catheter and vessel wall and facilitate intravascular manipulations during catheterization and placement of drug-eluting stents. One newly reported complication of such hydrophilic coatings is the fragmentation and embolization of the polymer, which can lead to ischemia and infarct by blocking the small vasculature. In this report, we present a patient with a non-healing ulcer on the leg. Biopsy from the ulcer revealed the presence of hydrophilic polymer emboli within the dermal vessels. This is one of the few reports of such a complication involving the skin. Our objective is to emphasize the histopathologic features of this uncommon iatrogenic phenomenon.

Sex, genetics, and the control of thyroxine and thyrotropin in mice.

BACKGROUND: Previously, we studied the genetic basis for variability in total thyroxine (TT4) as part of investigating induced Graves' hyperthyroidism in panels of genetically diverse recombinant inbred (RI) mice. Because Graves' disease occurs predominantly in women, we used female mice. A limitation of this approach is that thyrotropin (TSH) is undetectable by some assays in most female mice. METHOD: Variation in levels of serum TSH, TT4, and free thyroxine (FT4) was measured in males from three related RI families (CXB, BXH, and AXBXA) followed by quantitative genetic analysis and mapping of these traits. RESULTS: In general, TSH levels were higher in males than females. FT4 levels were also higher in males than in females, but TT4 sex differences were absent or inconsistent. Chromosomal linkage was only observed for TSH in BXH males and for FT4 in AXBXA males. Different chromosomes were linked to TT4 in males of the three RI sets. The most striking finding came from genetic linkages in males versus our previous data for females. TT4 was linked to the same chromosomal loci in CXB males and females. In contrast, TT4, FT4, and TSH were linked to different "sex-specific chromosomes" in AXBXA and BXH families. CONCLUSIONS: In three RI mouse families, TSH and FT4 were significantly higher in males than females. Linkage analysis revealed chromosomal overlap for TT4 in males and females for one RI set but striking sex differences for TT4, FT4, and TSH linkage in two RI sets. Our findings provide a cautionary note: genetic linkage analysis of thyroid hormones traits in mice should be studied separately in males and females.

Genetic linkages for thyroxine released in response to thyrotropin stimulation in three sets of recombinant inbred mice provide evidence for shared and novel genes controlling thyroid function.

BACKGROUND: Graves' hyperthyroidism is induced by immunizing mice with adenovirus expressing the human thyrotropin (TSH)-receptor. Using families of recombinant-inbred mice, we previously discovered that genetic susceptibility to induced thyroid-stimulating antibodies and hyperthyroidism are linked to loci on different chromosomes, indicating a fundamental genetic difference in thyroid sensitivity to ligand stimulation. An approach to assess thyroid sensitivity involves challenging genetically diverse lines of mice with TSH and measuring the genotype/strain-specific increase in serum thyroxine (T4). METHODS: We investigated genetic susceptibility and genetic control of T4 stimulation by 10 mU bovine TSH in female mice of the CXB, BXH, and AXB/BXA strain families, all previously studied for induced Graves' hyperthyroidism. RESULTS: Before TSH injection, T4 levels must be suppressed by inhibiting endogenous TSH secretion. Three daily intraperitoneal L-triiodothyronine injections efficiently suppressed serum T4 in females of 50 of 51 recombinant inbred strains. T4 stimulation by TSH was more strongly linked in CXB and BXH sets, derived from parental strains with divergent T4 stimulation, than in AXB/BXA strains generated from parents with similar TSH-induced responses. Genetic loci linked to the acute TSH-induced T4 response (hours) were not the same as those linked to induced hyperthyroidism (which develops over months). CONCLUSIONS: Genetic susceptibility for thyroid sensitivity to TSH stimulation was distinct for three families of inbred mouse lines. These observations parallel the human situation with multiple genetic loci contributing to the same trait and different loci associated with the same trait in different ethnic groups. Of the genetic loci highlighted in mice, three overlap with, or are located up or downstream, of human TSH-controlling genes. Other studies show that human disease genes can be identified through cross-species gene mapping of evolutionary conserved processes. Consequently, our findings suggest that novel thyroid function genes may yet be revealed in humans.

Probing structural variability at the N terminus of the TSH receptor with a murine monoclonal antibody that distinguishes between two receptor conformational forms.

Despite elucidation of the crystal structure of M22, a human thyroid-stimulating autoantibody (TSAb) bound to the TSH receptor (TSHR) leucine-rich repeat domain (LRD), the mechanism by which TSAs activate the TSHR and cause Graves' disease remains unknown. A nonstimulatory murine monoclonal antibody, 3BD10, and TSAb interact with the LRD N-terminal cysteine cluster and reciprocally distinguish between two different LRD conformational forms. To study this remarkable phenomenon, we investigated properties of 3BD10, which has a linear epitopic component. By synthetic peptide ELISA, we identified 3BD10 binding to TSHR amino acids E34, E35, and D36 within TSHR cysteine-bonded loop 2 (C31-C41), which includes R38, the most N-terminal contact residue of TSAb M22. On flow cytometry, despite not contributing to the 3BD10 and M22 epitopes, chimeric substitution (but not deletion) of TSHR cysteine-bonded loop 1 (C24-C29) eliminated 3BD10 binding to the TSHR ectodomain (ECD) expressed on the cell surface, as found previously for TSAb including M22. Furthermore, 3BD10 did not recognize all cell surface TSHR ECDs, consistent with recognition of only one conformational receptor form. Reversion to wild-type of small components of the loop 1 chimeric substitution partially restored 3BD10 binding to the TSHR-ECD but not to synthetic peptides tested by ELISA. Molecular modeling supports the concept that modification of TSHR C-bonded loop 1 influences loop 2 conformation as well as LRD residues further downstream. In conclusion, the present study with mouse monoclonal antibody 3BD10 confirms TSHR conformational heterogeneity and suggests that the N-terminal cysteine cluster may contribute to this structural variability.

Insight into thyroid-stimulating autoantibody interaction with the thyrotropin receptor N-terminus based on mutagenesis and re-evaluation of ambiguity in this region of the receptor crystal structure.

BACKGROUND: Thyroid-stimulating autoantibodies (TSAb) bind to the thyrotropin receptor (TSHR) extracellular domain, or ectodomain (ECD), comprising a leucine-rich repeat domain (LRD) linked by a hinge region to the transmembrane domain (TMD). The LRD (residues 22-260; signal peptide 1-21) contains two disulfide-bonded loops at its N-terminus. In the crystal structure of the isolated LRD complexed with human TSAb monoclonal antibody (mAb) M22, N-terminal disulfide loop 1 (residues 22-30) could not be determined because of crystal disorder. Nevertheless, present crystal structure data are interpreted to exclude a role for the LRD N-terminal disulfide loops in the TSAb epitope(s), contradicting prior functional evidence of a role for these loops in TSAb function. MATERIALS AND METHODS: To re-examine this issue we studied two cell types expressing the TSHR with the extreme N-terminal loop 1 (residues 22-30) deleted: the TSHR ECD lacking the TMD and tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and the TSH holoreceptor containing the TMD. Because TSAb including M22 "see" the holoreceptor poorly relative to the TSHR ECD-GPI, we used the latter to examine the effect of deleting residues 22-30 on M22 binding by flow cytometry and the holoreceptor to test the effect of this deletion on the functional response to M22. RESULTS: Deletion of TSHR N-terminal loop 1 (residues 22-30) reduced the number of TSHR-ECD-GPI recognized by M22 relative to two TSHR mAb with epitopes far downstream of the LRD N-terminal loops. Relative to control mAb 2C11, M22 recognized only 60.4% of cell surface receptors (p = 0.02). In contrast to M22 binding to TSHR-ECD-GPI, in functional studies with the TSH holoreceptor, M22 stimulation of cAMP generation was unaltered by the loop 1 deletion. CONCLUSIONS: Our data support the concept that TSAb interact with the cysteine-rich N-terminus of the TSHR. Comparison of crystal structures of the same TSHR LRD in complex with TSAb M22 or blocking antibody K1-70 helps reconcile contradictory viewpoints. A difference between M22 interaction with the identical TSHR N-terminus expressed on the TSHR-ECD-GPI and holoreceptor suggests that crystallization of the TSHR LRD-M22 complex may not provide a complete understanding of the functional TSAb epitope(s) in Graves' disease.

Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function.

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261-412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening 'C-peptide' region. Experimentally, hinge region amino acids 317-366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function ('minimal hinge') are lacking. Using as a template the functionally normal TSHR with residues 317-366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307-371 deletion) was also tolerated without functional alteration, as was deletion of residues 303-366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377-384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303-366 (64 residues) or 307-371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.

Variable suppression of serum thyroxine in female mice of different inbred strains by triiodothyronine administered in drinking water.

BACKGROUND: Recombinant-inbred mouse strains differ in their susceptibility to Graves'-like hyperthyroidism induced by immunization with adenovirus expressing the human thyrotropin (TSH) receptor. Because one genetic component contributing to this susceptibility is altered thyroid sensitivity to TSH receptor agonist stimulation, we wished to quantify thyroid responsiveness to TSH. For such studies, it is necessary to suppress endogenous TSH by administering L-3,5,3'-triiodothyronine (L-T3), with the subsequent decrease in serum thyroxine (T4) reflecting endogenous TSH suppression. Our two objectives were to assess in different inbred strains of mice (i) the extent of serum T4 suppression after L-T3 administration and (ii) the magnitude of serum T4 increase induced by TSH. METHODS: Mice were tail-bled to establish baseline-serum T4 before L-T3 administration. We initially employed a protocol of L-T3-supplemented drinking water for 7 days. In subsequent experiments, we injected L-T3 intraperitoneally (i.p.) daily for 3 days. Mice were then injected i.p. with bovine TSH (10 mU) and euthanized 5 hours later. Serum T4 was assayed before L-T3 administration, and before and after TSH injection. In some experiments, serum T3 and estradiol were measured in pooled sera. RESULTS: Oral L-T3 (3 or 5 µg/mL) suppressed serum T4 levels by 26%-64% in female BALB/c mice but >95% in males. T4 suppression in female B6 mice ranged from 0% to 90%. In C3H mice, L-T3 at 3 µg/mL was ineffective but 5 µg/mL achieved >80% serum T4 reduction. Unlike inbred mice, in outbred CF1 mice the same protocol was more effective: 83% in females and 100% suppression in males. The degree of T4 suppression was unrelated to baseline T4, T3, or estradiol, but was related to mouse weight and postmortem T3, with greater suppression in larger mice (outbred CF1 animals and inbred males). Among females with serum T4 suppression >80%, the increase in serum T4 after TSH injection was greater for BALB/c and C3H versus B6 mice. Moreover, the T4 increment was higher in female than in male BALB/c. CONCLUSIONS: Our data provide important, practical information for future in vivo studies in inbred mice: we recommend that responses to TSH be performed in female animals injected with L-T3 i.p. to suppress baseline T4.

Antibodies to thyroid peroxidase arise spontaneously with age in NOD.H-2h4 mice and appear after thyroglobulin antibodies.

Hashimoto's thyroiditis, a common autoimmune disease, is associated with autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). TPO, unlike abundant and easily purified Tg, is rarely investigated as an autoantigen in animals. We asked whether antibodies (Abs) develop to both TPO and Tg in thyroiditis that is induced (C57BL/6 and DBA/1 mice) or arises spontaneously (NOD.H-2h4 mice). Screening for TPOAbs was performed by flow cytometry using mouse TPO-expressing eukaryotic cells. Sera were also tested for binding to purified mouse Tg and human TPO. The antibody data were compared with the extent of thyroiditis. Immunization with mouse TPO adenovirus broke self-tolerance to this protein in C57BL/6 mice, but thyroiditis was minimal and TgAbs were absent. In DBA/1 mice with extensive granulomatous thyroiditis induced by Tg immunization, TPOAbs were virtually absent despite high levels of TgAbs. In contrast, antibodies to mouse TPO, with minimal cross-reactivity with human TPO, arose spontaneously in older (7-12 months) NOD.H-2h4 mice. Unexpectedly, TgAbs preceded TPOAbs, a time course paralleled in relatives of probands with juvenile Hashimoto's thyroiditis. These findings demonstrate a novel aspect of murine and human thyroid autoimmunity, namely breaking B cell self-tolerance occurs first for Tg and subsequently for TPO.

Short term effects of spironolactone on blood lipid profile: a 3-month study on a cohort of young women with hirsutism.

AIMS: To investigate the effects of spironolactone on serum lipids in women with hirsutism over a 3-month period. METHODS: In a prospective setting, 27 hirsute women (20 with polycystic ovary syndrome and seven with idiopathic hirsutism) with a mean age of 23.0 +/- 5.1 years were studied at baseline and 3 months after receiving a daily dose of 100 mg of spironolactone. Patients did not receive any other medications and did not go through a specific diet during the study. Lipid profile, fasting blood glucose, testosterone, dehydroepiandrosterone sulphate (DHEAS) and prolactin (PRL) were measured at baseline and 3 months after therapy. RESULTS: Mean body mass index of patients was 26.1 +/- 5.1 kg m(-2) before treatment and 25.9 +/- 5.7 kg m(-2) after treatment (NS). The therapy was associated with a significant decline of mean high-density lipoprotein (HDL), 39.5 mg dl(-1)[95% confidence interval (CI) 35.6, 43.4]vs. 32.2 mg dl(-1) (95% CI 29.2, 35.2), and a significant increase in mean low-density lipoprotein (LDL), 133.1 mg dl(-1) (95% CI 120.2, 146) vs. 150.8 mg dl(-1) (95% CI 139.1, 162.5), and cholesterol/HDL ratio, 5 (95% CI 4.4, 5.6) vs. 6.4 (95% CI 5.7, 7.1) (P < 0.05). No significant change was noted in total cholesterol, triglyceride or fasting blood glucose levels. Serum values of testosterone, DHEAS and PRL decreased significantly after 3 months of therapy (P < 0.05). CONCLUSIONS: Spironolactone might have adverse effects on serum lipoprotein levels by increasing LDL and decreasing HDL over a short course of treatment. While treating hirsutism with spironolactone, special care should be given to women with metabolic disorders such as dyslipidaemia.

Albuminuria and its correlates in an Iranian type 2 diabetic population.

OBJECTIVE: To study the prevalence and correlates of increased urinary albumin excretion (UAE) in an Iranian type 2 diabetic population. METHODS: Over a one year period since October 2002, 400 consecutive type 2 diabetic patients referred to an outpatient diabetes clinic, were enrolled in a cross sectional study. Subjects had no history of renal impairment or overt proteinuria. Data concerning demographic characteristics and cardiovascular risk factors were recorded and height, weight and blood pressure were measured. Glucose, cholesterol, HDL-C, LDL-C, triglyceride, apoprotein B, lipoprotein a, creatinine, and HbA1c were measured in fasting blood samples. Overnight twelve-hour UAE were assessed by immunoturbidometry method. Regression analyses were employed to determine the correlates of UAE. RESULTS: Out of 400 patients, 156 (40%) subjects had increased UAE (UAE > or = 30 mg/24 hour). The UAE was higher in males compared to females (145.5 vs. 72.1 mg/day; p < 0.05); however, the age and HDL adjusted UAE levels were not significantly different between men and women (120.1 vs. and 87.9 mg/day; p = 0.37). Increased UAE was correlated with decreasing HDL-C and a longer duration of diabetes independent of other variables; increased UAE was correlated with HbA1c as well. Age, systolic and diastolic blood pressure, total cholesterol, LDL-C, triglyceride, apoprotein B, lipoprotein a, and GFR did not correlate with increased UAE. CONCLUSION: In this study, increased UAE was considerably frequent among type 2 diabetic patients without any significant history of renal dysfunction. Albuminuria was found to be associated with dyslipidemia (low HDL-C), long duration of diabetes, and uncontrolled glycemia revealed by higher HbA1c.

Thyroid autoimmunity and recurrent spontaneous abortion in Iran: a case-control study.

OBJECTIVE: To determine the association of thyroglobulin antibodies (TG-Ab) and thyroid peroxidase antibodies (TPO-Ab) with recurrent spontaneous abortion in a euthyroid, nonpregnant population of women in Iran. METHODS: In this case-control study conducted between November 2003 and September 2006 in Tehran, Iran, nonpregnant women with a history of 3 or more consecutive pregnancy losses and age-matched, healthy parous women without a history of reproductive problems were assessed. Thyroid function tests were performed, which included assessment of thyroid-stimulating hormone, triiodothyronine, thyroxine, and the presence of TG-Ab and TPO-Ab. RESULTS: A total of 641 patients and 269 controls were included. Mean age (+/- SD) was 30.6 +/- 6.4 years (range, 16-51 years) in the patient group and 30.05 +/- 6.6 years (range, 18-48 years) in the control group. Thyroid antibodies were present in 157 of 641 patients (24.5%) and in 34 of 269 controls (12.6%) (P<.001). The presence of thyroid antibodies was significantly associated with recurrent abortion independent of the impact of age with an odds ratio of 2.24 (95% confidence interval, 1.5-3.35). CONCLUSIONS: In this population of women in Iran, TG-Ab and TPO-Ab were identified more frequently in women with recurrent abortions compared with controls, and thyroid autoimmunity was independently associated with a higher risk of recurrent abortion.