Evaluation of Genetic or Cellular Impairments in Type I IFN Immunity in a Cohort of Young Adults with Critical COVID-19.

Several genetic and immunological risk factors for severe COVID-19 have been identified, with monogenic conditions relating to 13 genes of type I interferon (IFN) immunity proposed to explain 4.8% of critical cases. However, previous cohorts have been clinically heterogeneous and were not subjected to thorough genetic and immunological analyses. We therefore aimed to systematically investigate the prevalence of rare genetic variants causing inborn errors of immunity (IEI) and functionally interrogate the type I IFN pathway in young adults that suffered from critical COVID-19 yet lacked comorbidities. We selected and clinically characterized a cohort of 38 previously healthy individuals under 50 years of age who were treated in intensive care units due to critical COVID-19. Blood samples were collected after convalescence. Two patients had IFN-α autoantibodies. Genome sequencing revealed very rare variants in the type I IFN pathway in 31.6% of the patients, which was similar to controls. Analyses of cryopreserved leukocytes did not indicate any defect in plasmacytoid dendritic cell sensing of TLR7 and TLR9 agonists in patients carrying variants in these pathways. However, lymphocyte STAT phosphorylation and protein upregulation upon IFN-α stimulation revealed three possible cases of impaired type I IFN signaling in carriers of rare variants. Together, our results suggest a strategy of functional screening followed by genome analyses and biochemical validation to uncover undiagnosed causes of critical COVID-19.

Bone replaces articular cartilage in the rat knee joint after prolonged immobilization.

BACKGROUND: Lost joint range of motion (ROM) is common in chronic osteoarthritis, alters regional weight-bearing across the articular surfaces, and contributes to loss of cartilage and bone alterations. Limited data exist on the regional effects on joints subjected to chronic losses of ROM. OBJECTIVE: To characterize the regional replacement by bone as part of articular cartilage degeneration after prolonged immobilization. METHODS: Eleven rat knees were rigidly-immobilized in flexion for 32weeks with contralateral and sham-operated (n=6) knees as controls. Sagittal medial tibial epiphysis histological sections assessed the anterior (non-weight-bearing), middle and posterior (both weight-bearing) regions. We quantified the distribution of collagen I, collagen II, cartilage thickness, glycosaminoglycan (GAG) staining, Mankin scoring, and subchondral bone plate cross-sectional area. Using immunohistochemistry (IHC), we visualized blood vessels, osteoblasts, and mesenchymal stem cells (MSCs). RESULTS: Immobilized cartilage had increased collagen I content in the anterior tibial region with picrosirius red staining (immobilized=61±20%; contralateral=43±12%, p=0.033; sham=20±10%, p=0.028) and collagen I IHC (immobilized=40±10%; contralateral=11±4%, p=0.003; sham=5±3%, p=0.043). Articular cartilage was thinner anteriorly (18±30µm) in immobilized knees versus contralateral (124±40µm, p<0.001) and sham (125±43µm, p=0.043). GAG staining covered 2±4% of the anterior articular area in immobilized knees versus 28±12% contralaterally (p=0.003) and 26±7% in sham (p=0.043). Mankin scores in immobilized knees were 4.7±1.7 versus 0.2±0.4 and 0±0 for contralateral and sham (p=0.003, p=0.042), respectively. The trabecular bone plate area of anterior and posterior regions showed relative loss of cross-sectional area in immobilized knees compared to controls (immobilized/contralateral area ratios of 0.67 and 0.46 respectively, both p=0.003), while the area in the middle region was preserved. Movat's pentachrome stain and CD31 staining showed chondral vascular ingrowth from subchondral bone. Osteocalcin and CD90 MSC staining were decreased in immobilized knees versus contralateral (p=0.003, p=0.036 respectively). CONCLUSIONS: Bony replacement characterizes articular cartilage degeneration of knees immobilized for 32weeks in the anterior, non-weight bearing region of the tibia. Replacement of cartilage by bone may have been mediated by chondral vascularization, suggesting irreversible changes. These findings stress the importance of weight-bearing and joint motion to maintain cartilage structure.

Imported leprosy in Italy.

BACKGROUND: Leprosy is far from being eliminated with more than 200,000 new cases detected (NCD)/year. OBJECTIVE: Retrospective analysis between 2003 and 2009 to compare the New Case Detected Rate (NCDR) observed in Italy in the immigrant population with the NCDR of the same population in their country of origin to verify if the cases observed are those expected or not. METHODS: Leprosy statistics were retrieved from the Italian leprosy register and from official WHO data. RESULTS: The NCD in Italy were lower than expected, from 2003 when the expected number of NCD was 40.5 between the legally resident immigrants, but only one case was diagnosed (98% of lower from the expected), to 2009 when four NCD were diagnosed and 41 were expected (90% lower from expected). CONCLUSIONS: This study points out a discrepancy between the observed and the expected cases of leprosy in Italy. Specifically, the number of NCD was less than expected for each studied year. Of course our data do not represent a validation, but only an indication of the leprosy diagnosis in Italy. Difficulty in accessing the health systems, fear of segregation, ignorance and illegal immigrant status with consequent fear of police arrest are possible explaining factors. The critical issue anyhow is the medical expertise. The role of the dermatologist is fundamental. For these reasons, there is still a need for wide spread leprosy teaching programmes. Although with few limitations, this study represents a first approach to validate the accuracy in leprosy diagnosis in Italy.

Immunophenotype of skin lymphocytic infiltrate in patients co-infected with Mycobacterium leprae and human immunodeficiency virus: a scenario dependent on CD8+ and/or CD20+ cells.

BACKGROUND: Leprosy occurs rarely in human immunodeficiency virus (HIV)-positive patients. In contrast to tuberculosis, there has been no report to date of an increase in HIV prevalence among patients with leprosy or of differences in leprosy's clinical spectrum. While several studies describe the systemic immune response profile in patients co-infected with HIV and leprosy, the local immune skin response has been evaluated in only a small number of case reports and limited series of patients. OBJECTIVE: To investigate the interaction between Mycobacterium leprae and HIV infection in the skin. METHODS: We investigated the presence and frequency of cells positive for CD4, CD8, CD20, TIA-1, FOXP3 and CD123 in lymphocytic infiltrates from 16 skin biopsies taken from 15 patients with HIV-leprosy co-infection. RESULTS: CD4+ cells were absent in infiltrates from 6 (38%) skin biopsies and present in 10 (62%) cases at low levels (<1·16%) of the lymphocytic infiltrate. CD8+ was the predominant phenotype in the infiltrate (99·4%), followed by TIA-1, expressed by >75% of CD8+ cells. FOXP3+ cells were also present, representing 3·4% of the lymphocytic infiltrate. CD20+ cells were detected in 75% of the cases; however, in two cases (12%) these cells represented 25-50% of the infiltrate, while in the other 10 cases (62%) they were present only focally (<25% of the infiltrate). CD123+ cells were not observed in any of the studied specimens. CONCLUSIONS: Data presented here suggest that cell-mediated immune responses to M. leprae are preserved at the site of disease and that in the absence of CD4+ cells, CD8+FOXP3+ and CD20+ cells may be involved in granuloma formation.

Mobile teledermatology for skin tumour screening: diagnostic accuracy of clinical and dermoscopic image tele-evaluation using cellular phones.

BACKGROUND: The ability to diagnose malignant skin tumours accurately and to distinguish them from benign lesions is vital in ensuring appropriate patient management. Little is known about the effects of mobile teledermatology services on diagnostic accuracy and their appropriateness for skin tumour surveillance. OBJECTIVE: To evaluate the diagnostic accuracy of clinical and dermoscopic image tele-evaluation for mobile skin tumour screening. METHODS: Over a 3-month period up to three clinical and dermoscopic images were obtained of 113 skin tumours from 88 patients using a mobile phone camera. Dermoscopic images were taken with a dermatoscope applied to the camera lens. Clinical and dermoscopic images of each lesion together with clinical information were separately teletransmitted for decision-making. Results were compared with those obtained by face-to-face examination and histopathology as the gold standard. RESULTS: A total of 322 clinical and 278 dermoscopic images were acquired; two (1%) clinical and 18 (6%) dermoscopic pictures were inadequate for decision-making. After excluding inadequate images, the majority of which were dermoscopic pictures, only 104 of the 113 skin tumours from 80 of 88 patients could be tele-evaluated. Among these 104 lesions, 25 (24%) benign nonmelanocytic, 15 (14%) benign melanocytic, 58 (56%) malignant nonmelanocytic and six (6%) malignant melanocytic lesions were identified. Clinical and dermoscopic tele-evaluations demonstrated strong concordance with the gold standard (κ = 0·84 for each) and similar high sensitivity and specificity for all diagnostic categories. With regard to the detailed diagnoses, clinical image tele-evaluation was superior to teledermoscopy resulting in 16 vs. 22 discordant cases. CONCLUSIONS: Clinical image tele-evaluation might be the method of choice for mobile tumour screening.

Establishment of a model of cortical bone repair in mice.

A model of cortical bone repair has been established for use in mice. The cortical defect consisted of a hole drilled through the entire diameter of the tibial diaphysis. The hematoma that initially filled the drill site was invaded by cells of mesenchymal appearance within 5 days of injury. Trabeculae of mineralized woven bone were present throughout the drill site by day 9. A reaction in the periosteum adjacent to the drill site, consisting of both new bone and cartilage formation, preceded deposition of bone tissue in the drill site. New woven bone was modeled to restore the marrow cavity to normal by 4 weeks after injury, and almost normal cortical structure was achieved by 6 weeks after injury. Immunohistochemical studies indicated that type III collagen was expressed within the drill site by day 5, reached a peak at day 7, and was diminished by day 9. In contrast, type I collagen was first detectable in the drill site at day 7, and staining was more intense by day 9. Osteopontin expression in the drill site coincided with the process of mineralization of new bone in this location. The model of bone repair described here provides a method for inducing reproducible bone lesions in a readily identifiable location in mice. It will be useful in the investigation of bone cell function in mouse strains that have been subjected to genetic manipulation.

Rat middle cerebral artery occlusion: correlations between histopathology, T2-weighted magnetic resonance imaging, and behavioral indices.

During attempts to develop the intraluminal suture model of transient middle cerebral artery occlusion (MCAO) in the Sprague Dawley strain of rats, we noticed a wide variability in lesion size seen with T2-weighed magnetic resonance imaging (MRI) or histopathology, as well as in scores for behavioral indices. It was our intent to examine the results of the study carefully and determine whether there were strong point-to-point correlations between the degree of lesion size determined from T2-weighted MRI or histopathology and intermediate or long-term neurologic/behavioral assessments. Baseline behavioral scores for forelimb dexterity (staircase test) were obtained on all animals in the period before receiving 60 minutes of transient MCAO. After MCAO, animals were tested at specified intervals from 1 to 21 days for composite neurologic deficits. T2-weighted MRI was taken at 2 and 7 days post-MCAO. At 30 and 60 days post-MCAO, animals were retested in the staircase test with subsequent histopathologic examination of the brains. Indeed, there were highly significant correlations between lesion size determined by MRI and histopathology. The damage observed in the T2-weighted MRI, as well as the size of the histopathologic lesions, were in turn highly correlated to deficiencies observed in the composite neurologic assessments, as well as to deficits at 30 and 60 days post-MCAO for skilled use of the contralateral forepaw (damaged side). In the latter test, the correlations were somewhat less significant for the ability of rats to reach for food with the ipsilateral forepaw (undamaged side).

Effect of fucoidan treatment on collagenase-induced intracerebral hemorrhage in rats.

Inflammatory cells are postulated to mediate some of the brain damage following ischemic stroke. Intracerebral hemorrhage is associated with more inflammation than ischemic stroke. We tested the sulfated polysaccharide fucoidan, which has been reported to reduce inflammatory brain damage, in a rat model of intracerebral hemorrhage induced by injection of bacterial collagenase into the caudate nucleus. Rats were treated with seven day intravenous infusion of fucoidan (30 micrograms h-1) or vehicle. The hematoma was assessed in vivo by magnetic resonance imaging. Motor behavior, passive avoidance, and skilled forelimb function were tested repeatedly for six weeks. Fucoidan-treated rats exhibited evidence of impaired blood clotting and hemodilution, had larger hematomas, and tended to have less inflammation in the vicinity of the hematoma after three days. They showed significantly more rapid improvement of motor function in the first week following hemorrhage and better memory retention in the passive avoidance test. Acute white matter edema and eventual neuronal loss in the striatum adjacent to the hematoma did not differ between the two groups. Investigation of more specific anti-inflammatory agents and hemodiluting agents are warranted in intracerebral hemorrhage.

-(S)-Alpha-phenyl-2-pyridine-ethanamine Dihydrochloride-, a low affinity uncompetitive N-methyl-D-aspartic acid antagonist, is effective in rodent models of global and focal ischemia.

[(S)-Alpha-phenyl-2-pyridine-ethanamine dihydrochloride] (ARL 15896AR) is a low affinity uncompetitive N-methyl-D-aspartic acid receptor antagonist that was tested in animal models of anoxia and ischemia. Pretreatment of rodents with ARL 15896AR extended survival time during exposure to hypoxia. With the rat four-vessel occlusion model of global ischemia (20 min), oral dosing commencing at reflow, resulted in significant protection of the CA1 hippocampal neurons. ARL 15896AR was, however, ineffective in the rat two-vessel occlusion model and in the gerbil models of forebrain ischemia, the latter due to an inability to attain suitable plasma levels. In the spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) (2 hr plus 22 hr reflow), acute dosing with ARL 15896AR (i.p.) beginning from 30 min before or up to 1 hr post-MCAO significantly reduced cortical infarct volume. The ability of ARL 15896AR to influence infarct size, as well as functional correlates was examined in SHR after 90 min of MCAO. T2 weighted magnetic resonance images taken at 2 and 6 days post-MCAO revealed significantly smaller lesion sizes in the group receiving injections with ARL 15896AR beginning 30 min after occlusion. Spontaneously hypertensive rats were subsequently tested (30-42 days post-MCAO) and found to be deficient in skilled use of the forepaws (staircase test). The contralateral forepaw was most severely impaired, however, ARL 15896AR treatment prevented motor impairment in only the ipsilateral forepaw. Histopathological examination of cortical infarct size was unremarkable between treated and control rats. The findings indicate that ARL 15896AR exhibits neuroprotection in global and focal models of ischemia

A vinyl polysiloxane die used to make interim restorations.

A die made of vinyl polysiloxane is a great aid for making interim restorations and for establishing correct gingival contours for finer restorations. This article describes the rationale and procedures for such a procedure.

Hyperleukocytosis in leukemia.

Hyperleukocytosis in leukemia challenges physicians and nurses to provide swift and aggressive care to the patient so that complications from leukostasis are avoided. It is therefore prudent for nurses to recognize patients at risk for hyperleukocytosis. Prevention of complications is the key because once pulmonary and central nervous system symptoms arise the probability for reversal becomes uncertain. The major role of nurses caring for patients with hyperleukocytosis is to provide baseline assessment of pulmonary and neurological function, with continuous follow-up assessment to detect early signs and symptoms of organ dysfunction. Once complications from hyperleukocytosis are apparent, nursing interventions are aimed at easing respiratory distress, minimizing increases in intercranial pressure, and providing emotional support for the patient and his family.

Nodular pulmonary amyloidosis.

An elderly man had a 10-year history of multiple pulmonary nodules that he had refused to have investigated. He died of a ruptured abdominal aortic aneurysm. At autopsy the nodules were shown to consist of amyloid. There was no evidence of systemic amyloidosis.