Search

Digital pathology for the routine assessment of cases for primary diagnosis has been implemented by few laboratories worldwide. The Gravina Hospital in Caltagirone (Sicily, Italy), which collects cases from 7 different hospitals distributed in the Catania area, converted the entire workflow to digital starting from 2019. Before the transition, the Caltagirone pathology laboratory was characterized by a non-tracked workflow, based on paper requests, hand-written blocks and slides, as well as manual assembling and delivering of the cases and glass slides to the pathologists. Moreover, the arrangement of the spaces and offices in the department was illogical and under-productive for the linearity of the workflow. For these reasons, an adequate 2D barcode system for tracking purposes, the redistribution of the spaces inside the laboratory and the implementation of the whole-slide imaging (WSI) technology based on a laboratory information system (LIS)-centric approach were adopted as a needed prerequisite to switch to a digital workflow. The adoption of a dedicated connection for transfer of clinical and administrative data between different software and interfaces using an internationally recognised standard (Health Level 7, HL7) in the pathology department further facilitated the transition, helping in the integration of the LIS with WSI scanners. As per previous reports, the components and devices chosen for the pathologists' workstations did not significantly impact on the WSI-based reporting phase in primary histological diagnosis. An analysis of all the steps of this transition has been made retrospectively to provide a useful "handy" guide to lead the digital transition of "analog", non-tracked pathology laboratories following the experience of the Caltagirone pathology department. Following the step-by-step instructions, the implementation of a paperless routine with more standardized and safe processes, the possibility to manage the priority of the cases and to implement artificial intelligence (AI) tools are no more an utopia for every "analog" pathology department.

We previously identified a group of long-term pediatric survivors who had acquired HIV-1 through maternal transmission; had not received antiretroviral therapy; are now >8 years old, in good health, and with no opportunistic infections; and have not failed to thrive, although they have greatly decreased numbers of blood CD4+ T cells (<500/mm(3)). All the children have elevated total serum IgE levels (210-2475 IU/ml) and make anti-HIV-1 IgE or IgE directed against non-HIV-1 specificities (radioimmunoassay, Western blot assay); they have no detectable antigenemia. We have now studied the ability of anti-HIV-1 IgE in serum obtained from these children to regulate (1) production of HIV-1 by interleukin 2/phytohemagglutinin (IL-2/PHA)-stimulated peripheral blood mononuclear cells (PBMCs) taken from HIV-1-seronegative donors and infected with a T cell-tropic clone of HIV-1, and (2) transmission of a primary HIV-1 strain from adult AIDS patients to uninfected IL-2/PHA-stimulated PBMCs (p24 core antigen production). High levels of HIV-1 production were observed when PBMCs were cultured for 5 days in the presence of HIV-1-seronegative donor serum that was either IgE positive or IgE negative (IgE, >100 or <100 IU/ml, respectively). HIV-1 production also was observed when PBMCs were cultured with HIV-1-infected donor serum that either contained IgE directed against non-HIV-1 specificities or was IgE negative; these levels were 40% less than those seen with sera from the HIV-1-seronegative donors. Far greater inhibition of virus production was observed if the serum in culture contained anti-HIV-1 IgE (>95%). Virus neutralization did not appear to account for the inhibition obtained with anti-HIV-1 IgE-containing serum because virus production was not suppressed in cultures to which serum was added immediately preinfection (<10%), but was strongly suppressed when serum was added 1.5 hr postinfection (>95%). The inhibition of virus production obtained with serum containing anti-HIV-1 IgE was reversed when (1) serum was depleted of IgE (immunoaffinity), but not when it was depleted of IgG (protein G-Sepharose) before inclusion in culture postinfection, (2) anti-IgE, but not anti-IgG, was included in culture, or (3) serum was heat treated before culture. The results indicate that serum from certain HIV-1-infected pediatric long-term survivors contains agents that inhibit HIV-1 production in vitro, and that these agents include anti-HIV-1 IgE. They suggest that a cytotoxic event, rather than virus neutralization, plays an important role in anti-HIV-1 IgE-mediated inhibition of virus production.

Pathophysiological mechanisms of primary headache remain obscure, despite of numerous hypotheses that have been postulated for either migraine and cluster headache. Human experimental models are not available, however, observation of clinical features of migraine or cluster headache attacks support animal studies documenting the development of neurogenic inflammation in tissues receiving trigeminal innervation. The latter studies provided also the background for better understanding the mechanism of action of aborting drugs such as sumatriptan and dihydroergotamine. The debate is whether the primary cause of migraine and other neurovascular headaches is central or peripheral in origin. Trigger factors (stressful events) and personality traits in migraine patients suggest that activation of neurovascular systems is secondary to more complex events taking place in the central nervous system.

Pathophysiological mechanisms of primary headache remain obscure, despite of numerous hypotheses that have been postulated for either migraine and cluster headache. Human experimental models are not available, however, observation of clinical features of migraine or cluster headache attacks support animal studies documenting the development of neurogenic inflammation in tissues receiving trigeminal innervation. The latter studies provided also the background for better understanding the mechanism of action of aborting drugs such as sumatriptan and dihydroergotamine. The debate is whether the primary cause of migraine and other neurovascular headaches is central or peripheral in origin. Trigger factors (stressful events) and personality traits in migraine patients suggest that activation of neurovascular systems is secondary to more complex events taking place in the central nervous system.

We have previously shown that during human immunodeficiency virus type 1 (HIV-1) infection in vitro continued reverse transcription is required for stable HIV-1 production, but entry by progeny virus is not. To determine the source of the viral RNA reverse-transcribed late in infection, we employed inhibitors of HIV-1 transmission, reverse transcription, and proteolysis of the Gag-Pol polyprotein to interrupt HIV-1 infection in vitro. The kinetics of synthesis of viral DNA, RNA, and proteins was examined. During single-cycle infection, inhibition of reverse transcription 24-72 hr after infection delayed production of viral RNA and protein 10 days. Although viral DNA was detected in Southern blots, inhibition of Gag-Pol processing or transient inhibition of reverse transcription blocked its expression. We propose that after initial reverse transcription of input virion RNA is complete, newly synthesized HIV-1 RNA is reverse-transcribed before its export in virions to yield the viral DNA required for stable HIV-1 production.

Pulmonary cells and fluid obtained by bronchoalveolar lavage (BAL) from 19 pediatric acquired immunodeficiency syndrome (AIDS) patients with pneumonia were examined for markers of human immunodeficiency virus (HIV-1) infection. The HIV-1 DNA was detected in BAL cells by polymerase chain reaction (PCR) in 14 of 15 patients (93.3 percent). Immunostaining of cytocentrifuged cell preparations of six specimens revealed that HIV-1 antigen was associated with from five percent to 95 percent of the alveolar macrophages. Analysis of the 22 cell-free BAL fluids by enzyme immunoassay (EIA) showed that samples from three patients (15.8 percent) contained HIV-1 p24 antigen. One sample, with a dilution factor of 15.1 relative to serum, contained a markedly elevated antigen concentration (106 pg per ml) compared to the serum concentration (41.6 pg per ml). Antibodies to HIV-1 were present in the BAL fluids of six patients (31.6 percent) at levels detectable by EIA. By Western blot analysis, three samples yielded more intense gp120 bands compared to bands observed with matched serum samples. Our results suggest that HIV-1 and antibodies to this virus are frequently present in the lungs of children with AIDS and that the serum antigen and antibody profile of some patients does not reflect local pulmonary levels.

Evaluation of the activities of antiretroviral agents and an immunoregulatory compound has been made using two models of HIV-1 infection and three measurements of virus expression. Acute infection of Jurkat cells or chronic/inducible infection in U1.1 cells was monitored at multiple time points after drug treatment. The 50% effective concentrations (EC50) of the HIV-1 inhibitors suramin, 3'-azido-3'-deoxythymidine (AZT), and 2',3'-dideoxycytidine, as measured by HIV-1 RNA hybridization in Jurkat cells two days after infection, were comparable to EC50 values obtained in parallel measurements of extracellular p24 levels and percent HIV-1 IF-positive cells. However, these measurements diverged: at seven days after infection the EC50 of AZT was greater than 10 microM when intracellular HIV-1 RNA was assayed, 0.2 microM by IF, and 0.03 microM by p24 assay. Human thymic humoral factor displayed no direct inductive activity in chronic HIV-1 infection in U1.1 cells, while phorbol ester and lymphocyte supernatants induced all parameters. These observations warrant care when interpreting results of only a single assay and suggest that definitive assay of HIV-1 infection requires measurements of multiple parameters of virus expression.

The role of local immunity in relation to the frequent and heterogeneous pulmonary manifestations of HIV-1 infection in children is poorly understood. In order to examine lung immunity in pediatric AIDS patients, the cellular composition, immunoglobulin, and immune complex (IC) levels were evaluated in 23 samples of bronchoalveolar lavage (BAL) fluid and peripheral blood from 19 pediatric AIDS patients with acute pulmonary pathology. The patients were of two age groups: 4.0-21.5 months (N = 9) and 2.3-13.1 years (N = 10). In BAL, lymphocytes were elevated in 25-45% of samples, and neutrophils were elevated in 27-33%; BAL macrophages varied in percentage (28-99%) but had normal morphology. The blood differentials of pediatric AIDS patients undergoing BAL did not show significant differences when compared with a group of pediatric patients with tuberculosis, but leuko- and neutropenia was noted when compared with pediatric patients with pneumonia and no HIV disease. Of the immunoglobulins measured (IgG, IgM, IgA) only IgG was detectable in unconcentrated BAL fluid (1-37 mg/dl, equivalent to 12-630 mg/dl in the epithelial lining fluid after correction using urea as a marker of dilution). All patients were hypergammaglobulinemic and 83% had high levels of circulating IC (2-40 muEq/ml). Six BAL specimens (26%) also contained IC. The estimated level of IC in lung epithelial lining fluid (after correcting for dilution) was up to fivefold higher than IC concentration in corresponding sera. Specific antibodies to HIV-1 were demonstrated in 35% of the BAL samples by ELISA and in 65% by Western blotting.(ABSTRACT TRUNCATED AT 250 WORDS)

Human immunodeficiency virus 1 (HIV-1) infection in vitro has been analyzed by the kinetics of expression of HIV-1 RNA and antigens during treatment with antiviral agents. Intracellular HIV-1 RNA rose from input values of 15 molecules per cell to 28 molecules per cell within 3 h after infection and reached a peak of 13,125 in 5 days. The first detectable increase in levels of HIV-1 capsid protein production was 1 day after infection. Virus infection was interrupted at different time points by the introduction of either 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, or suramin to block reverse transcription, or recombinant soluble CD4 to block binding and re-entry of progeny virus. Two results are noteworthy. First, the three inhibitors of reverse transcription blocked viral expression when added up to 48 h after infection. Second, the extent of infection, although postponed, is not greatly altered by culture of infected cells in recombinant soluble CD4. These data imply that reinfection with progeny virus, while necessary for rapid virus expression, is not required for the establishment of productive HIV-1 infection.

An assay is described for titration of human immunodeficiency virus type 1 (HIV-1) and for quantitative analysis of virus expression in vitro. The assay utilizes a liquid RNA-RNA hybridization method coupled with reversible target capture (RTC) on oligo(dT) derivatized magnetic particles. The assay provides a rapid, specific, and sensitive method for quantitation of HIV-1 RNA molecules present either in cells or in viral particles from cell-free culture media. Chronically infected monocytoid U1.1 cells were found to carry 52 pg HIV-1 RNA per 200,000 cells (160 HIV-1 RNA molecules per cell). In contrast, acutely infected CEM and H9 cells carried 3010 and 4370 pg HIV-1 RNA per 200,000 cells (9040 and 13,110 HIV-1 RNA molecules per cell, respectively). No hybridization was observed with uninfected cells or cells infected with HIV-2, HTLV-I, HTLV-II, or EBV. Use of liquid HIV-1 RNA hybridization in association with HIV-1 protein detection methods permits more complete characterization of HIV-1 expression in host cells than either method alone, and also provides a method for standardizing preparations of virus.

We report a case of a 36-year-old right-handed woman, who, in the 40th week of her first pregnancy, presented light coma which lasted 6 days. When the patient came out of coma she presented very marked oral tendency and hypermetamorphosis resembling the Kluver-Bucy syndrome in man. As sustained in the literature, the lesion showed by CT scan was bilateral and involved mainly the limbic structures in baso-temporal sites. The patient is compared to other cases described in literature.

The antiviral activity of mismatched dsRNA of the form poly(I):poly(C12-U)n (Ampligen) against the human immunodeficiency virus type 1 (HIV-1) was investigated by RNA-RNA and RNA-DNA hybridizations. Mismatched dsRNA delayed the appearance of newly transcribed HIV-1 RNA as detected by liquid dot-blot hybridization in cultures of H9 T-lymphoblastoid cells following virus challenge. The appearance of proviral DNA as detected by Southern hybridization following virus challenge in H9 cells was also delayed. Mismatched dsRNA had no effect in syncytium inhibition assays performed by fusing MT-2 cells with H9/HTLV-IIIB cells. These results suggest that the in vitro anti-HIV-1 activity of mismatched dsRNA occurs, at least in part, at an early stage in the viral replication cycle following initial gp120-CD4 binding.

10 patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), or lymphadenopathy syndrome (LAS) were given 200-250 mg ampligen, a mismatched double-stranded (ds) RNA with in-vitro antiviral activity against human immunodeficiency virus (HIV), twice a week for up to 18 weeks, without side-effects or toxicity. In all 9 patients who were positive for HIV RNA in peripheral blood mononuclear cells before therapy, levels became undetectable between days 10 and 40 of the start of therapy. 6 of the 7 patients with ARC or LAS also showed a progressive reduction in HIV load as measured by co-culture assays. All 10 patients had augmentation of delayed-type hypersensitivity skin reactions. Other changes noted during ampligen therapy included an increase in or maintenance of numbers of helper-inducer T lymphocytes, improvements in HIV-related symptoms, rises in titre of neutralising antibodies against HIV, and restoration of proper functioning of the natural lymphocyte antiviral dsRNA-dependent (2'-5'-oligoadenylate/RNA-ase L) pathway. Thus, in the short term, ampligen seems to have the dual ability to restore immunological function and to control HIV replication.