Clinical relevance of thymic and bone marrow outputs in multiple sclerosis patients treated with alemtuzumab.

Thymic and bone marrow outputs were evaluated in 13 sequential samples of 68 multiple sclerosis patients who initiated alemtuzumab and were clinically followed for 48 months. Three months after alemtuzumab infusions, the levels of new T lymphocytes were significantly reduced, but progressively increased reaching the highest values at 36 months, indicating the remarkable capacity of thymic function recovery. Newly produced B cells exceeded baseline levels as early as 3 months after alemtuzumab initiation. Heterogeneous patterns of new T- and B-cell recovery were identified, but without associations with age, sex, previous therapies, development of secondary autoimmunity or infections, and disease re-emergence. Trial registration version 2.0-27/01/2016.

Incidence and Long-term Functional Outcome of Neurologic Disorders in Hospitalized Patients With COVID-19 Infected With Pre-Omicron Variants.

BACKGROUND AND OBJECTIVES: A variety of neurologic disorders have been reported as presentations or complications of coronavirus disease 2019 (COVID-19) infection. The objective of this study was to determine their incidence dynamics and long-term functional outcome. METHODS: The Neuro-COVID Italy study was a multicenter, observational, cohort study with ambispective recruitment and prospective follow-up. Consecutive hospitalized patients presenting new neurologic disorders associated with COVID-19 infection (neuro-COVID), independently from respiratory severity, were systematically screened and actively recruited by neurology specialists in 38 centers in Italy and the Republic of San Marino. The primary outcomes were incidence of neuro-COVID cases during the first 70 weeks of the pandemic (March 2020-June 2021) and long-term functional outcome at 6 months, categorized as full recovery, mild symptoms, disabling symptoms, or death. RESULTS: Among 52,759 hospitalized patients with COVID-19, 1,865 patients presenting 2,881 new neurologic disorders associated with COVID-19 infection (neuro-COVID) were recruited. The incidence of neuro-COVID cases significantly declined over time, comparing the first 3 pandemic waves (8.4%, 95% CI 7.9-8.9; 5.0%, 95% CI 4.7-5.3; 3.3%, 95% CI 3.0-3.6, respectively; p = 0.027). The most frequent neurologic disorders were acute encephalopathy (25.2%), hyposmia-hypogeusia (20.2%), acute ischemic stroke (18.4%), and cognitive impairment (13.7%). The onset of neurologic disorders was more common in the prodromic phase (44.3%) or during the acute respiratory illness (40.9%), except for cognitive impairment whose onset prevailed during recovery (48.4%). A good functional outcome was achieved by most patients with neuro-COVID (64.6%) during follow-up (median 6.7 months), and the proportion of good outcome increased throughout the study period (r = 0.29, 95% CI 0.05-0.50; p = 0.019). Mild residual symptoms were frequently reported (28.1%) while disabling symptoms were common only in stroke survivors (47.6%). DISCUSSION: Incidence of COVID-associated neurologic disorders decreased during the prevaccination phase of the pandemic. Long-term functional outcome was favorable in most neuro-COVID disorders, although mild symptoms commonly lasted more than 6 months after infection.

Markers of blood-brain barrier disruption increase early and persistently in COVID-19 patients with neurological manifestations.

Background: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is associated with disorders affecting the peripheral and the central nervous system. A high number of patients develop post-COVID-19 syndrome with the persistence of a large spectrum of symptoms, including neurological, beyond 4 weeks after infection. Several potential mechanisms in the acute phase have been hypothesized, including damage of the blood-brain-barrier (BBB). We tested weather markers of BBB damage in association with markers of brain injury and systemic inflammation may help in identifying a blood signature for disease severity and neurological complications. Methods: Blood biomarkers of BBB disruption (MMP-9, GFAP), neuronal damage (NFL) and systemic inflammation (PPIA, IL-10, TNFα) were measured in two COVID-19 patient cohorts with high disease severity (ICUCovid; n=79) and with neurological complications (NeuroCovid; n=78), and in two control groups free from COVID-19 history, healthy subjects (n=20) and patients with amyotrophic lateral sclerosis (ALS; n=51). Samples from COVID-19 patients were collected during the first and the second wave of COVID-19 pandemic in Lombardy, Italy. Evaluations were done at acute and chronic phases of the COVID-19 infection. Results: Blood biomarkers of BBB disruption and neuronal damage are high in COVID-19 patients with levels similar to or higher than ALS. NeuroCovid patients display lower levels of the cytokine storm inducer PPIA but higher levels of MMP-9 than ICUCovid patients. There was evidence of different temporal dynamics in ICUCovid compared to NeuroCovid patients with PPIA and IL-10 showing the highest levels in ICUCovid patients at acute phase. On the contrary, MMP-9 was higher at acute phase in NeuroCovid patients, with a severity dependency in the long-term. We also found a clear severity dependency of NFL and GFAP levels, with deceased patients showing the highest levels. Discussion: The overall picture points to an increased risk for neurological complications in association with high levels of biomarkers of BBB disruption. Our observations may provide hints for therapeutic approaches mitigating BBB disruption to reduce the neurological damage in the acute phase and potential dysfunction in the long-term.

Neurologic manifestations in 1760 COVID-19 patients admitted to Papa Giovanni XXIII Hospital, Bergamo, Italy.

OBJECTIVES: Evidences from either small series or spontaneous reporting are accumulating that SARS-CoV-2 involves the Nervous Systems. The aim of this study is to provide an extensive overview on the major neurological complications in a large cohort of COVID-19 patients. METHODS: Retrospective, observational analysis on all COVID-19 patients admitted from February 23rd to April 30th, 2020 to ASST Papa Giovanni XXIII, Bergamo, Italy for whom a neurological consultation/neurophysiological assessment/neuroradiologic investigation was requested. Each identified neurologic complication was then classified into main neurologic categories. RESULTS: Of 1760 COVID-19 patients, 137 presented neurologic manifestations that manifested after COVID-19 symptoms in 98 pts and was the presenting symptom in 39. Neurological manifestations were classified as: (a) cerebrovascular disease [53 pts (38.7%)] including 37 ischemic and 11 haemorrhagic strokes, 4 transient ischemic attacks, 1 cerebral venous thrombosis; (b) peripheral nervous system diseases [31 (22.6%)] including 17 Guillain-Barrè syndromes; (c) altered mental status [49 (35.8%)] including one necrotizing encephalitis and 2 cases with RT-PCR detection of SARS-Cov-2 RNA in CSF; (d) miscellaneous disorders, among whom 2 patients with myelopathy associated with Ab anti-SARS-CoV-2 in CSF. Patients with peripheral nervous system involvement had more frequently severe ARDS compared to patients with cerebrovascular disease (87.1% vs 42%; difference = 45.1% 95% CI 42.0-48.2; χ2= 14.306; p < 0.0002) and with altered mental status (87.1% vs 55.6%; difference = 31.5% 95% CI 27.5-37.5%; χ2= 7.055; p < 0.01). CONCLUSION: This study confirms that involvement of nervous system is common in SARS-CoV-2 infection and offers clinicians useful information for prevention and prompt identification in order to set the adequate therapeutic strategies.

Altered methylation pattern of the SRD5A2 gene in the cerebrospinal fluid of post-finasteride patients: a pilot study.

CONTEXT: Post-finasteride syndrome (PFS) occurs in patients with androgenic alopecia after suspension of the finasteride treatment, leading to a large variety of persistent side effects. Despite the severity of the clinical picture, the mechanism underlying the PFS symptoms onset and persistence is still unclear. OBJECTIVE: To study whether epigenetic modifications occur in PFS patients. METHODS: Retrospective analysis of a multicentric, prospective, longitudinal, case-control clinical trial, enrolling 16 PFS patients, compared to 20 age-matched healthy men. Main outcomes were methylation pattern of SRD5A1 and SRD5A2 promoters and concentration of 11 neuroactive steroids, measured by liquid chromatography-tandem mass spectrometry, in blood and cerebrospinal fluid (CSF) samples. RESULTS: SRD5A1 and SRD5A2 methylation analysis was performed in all blood samples (n = 16 PFS patients and n = 20 controls), in 16 CSF samples from PFS patients and in 13 CSF samples from controls. The SRD5A2 promoter was more frequently methylated in CSF of PFS patients compared to controls (56.3 vs 7.7%). No promoter methylation was detected in blood samples in both groups. No methylation occurred in the SRD5A1 promoter of both groups. Unmethylated controls compared to unmethylated SRD5A2 patients showed higher pregnenolone, dihydrotestosterone and dihydroprogesterone, together with lower testosterone CSF levels. Andrological and neurological assessments did not differ between methylated and unmethylated subjects. CONCLUSIONS: For the first time, we demonstrate a tissue-specific methylation pattern of SRD5A2 promoter in PFS patients. Although we cannot conclude whether this pattern is prenatally established or induced by finasteride treatment, it could represent an important mechanism of neuroactive steroid levels and behavioural disturbances previously described in PFS.

Neuroactive steroid levels and psychiatric and andrological features in post-finasteride patients.

Recent reports show that, in patients treated with finasteride for male pattern hair loss, persistent side effects including sexual side effects, depression, anxiety and cognitive complaints may occur. We here explored the psychiatric and andrological features of patients affected by post-finasteride syndrome (PFS) and verified whether the cerebrospinal fluid (CSF) and plasma levels of neuroactive steroids (i.e., important regulators of nervous function) are modified. We found that eight out of sixteen PFS male patients considered suffered from a DSM-IV major depressive disorder (MDD). In addition, all PFS patients showed erectile dysfunction (ED); in particular, ten patients showed a severe and six a mild-moderate ED. We also reported abnormal somatosensory evoked potentials of the pudendal nerve in PFS patients with severe ED, the first objective evidence of a neuropathy involving peripheral neurogenic control of erection. Testicular volume by ultrasonography was normal in PFS patients. Data obtained on neuroactive steroid levels also indicate interesting features. Indeed, decreased levels of pregnenolone, progesterone and its metabolite (i.e., dihydroprogesterone), dihydrotestosterone and 17beta-estradiol and increased levels of dehydroepiandrosterone, testosterone and 5alpha-androstane-3alpha,17beta-diol were observed in CSF of PFS patients. Neuroactive steroid levels were also altered in plasma of PFS patients, however these changes did not reflect exactly what occurs in CSF. Finally, finasteride did not only affect, as expected, the levels of 5alpha-reduced metabolites of progesterone and testosterone, but also the further metabolites and precursors suggesting that this drug has broad consequence on neuroactive steroid levels of PFS patients.

Characteristics and Properties of Mesenchymal Stem Cells Derived From Microfragmented Adipose Tissue.

The subcutaneous adipose tissue provides a clear advantage over other mesenchymal stem cell sources due to the ease with which it can be accessed, as well as the ease of isolating the residing stem cells. Human adipose-derived stem cells (hADSCs), localized in the stromal-vascular portion, can be isolated ex vivo using a combination of washing steps and enzymatic digestion. In this study, we report that microfragmented human lipoaspirated adipose tissue is a better stem cell source compared to normal lipoaspirated tissue. The structural composition of microfragments is comparable to the original tissue. Differently, however, this procedure activates the expression of antigens, such as ß-tubulin III. The hADSCs derived from microfragmented lipoaspirate tissue were systematically characterized for growth features, phenotype, and multipotent differentiation potential. They fulfill the definition of mesenchymal stem cells, although with a higher neural phenotype profile. These cells also express genes that constitute the core circuitry of self-renewal such as OCT4, SOX2, and NANOG, and neurogenic lineage genes such as NEUROD1, PAX6, and SOX3. Such findings suggest further studies by evaluating Microfrag-AT hADSC action in animal models of neurodegenerative conditions.

Neuroactive steroid levels in plasma and cerebrospinal fluid of male multiple sclerosis patients.

Neuroactive steroid family includes molecules synthesized in peripheral glands (i.e., hormonal steroids) and directly in the nervous system (i.e., neurosteroids) which are key regulators of the nervous function. As already reported in clinical and experimental studies, neurodegenerative diseases affect the levels of neuroactive steroids. However, a careful analysis comparing the levels of these molecules in cerebrospinal fluid (CSF) and in plasma of multiple sclerosis (MS) patients is still missing. To this aim, the levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in CSF and plasma of male adults affected by Relapsing-Remitting MS and compared with those collected in control patients. An increase in pregnenolone and isopregnanolone levels associated with a decrease in progesterone metabolites, dihydroprogesterone, and tetrahydroprogesterone was observed in CSF of MS patients. Moreover, an increase of 5α-androstane-3α,17ß-diol and of 17ß-estradiol levels associated with a decrease of dihydrotestosterone also occurred. In plasma, an increase in pregnenolone, progesterone, and dihydrotestosterone and a decrease in dihydroprogesterone and tetrahydroprogesterone levels were reported. This study shows for the first time that the levels of several neuroactive steroids, and particularly those of progesterone and testosterone metabolites, are deeply affected in CSF of relapsing-remitting MS male patients. We here demonstrated that, the cerebrospinal fluid and plasma levels of several neuroactive steroids are modified in relapsing remitting multiple sclerosis male patients. Interestingly, we reported for the first time that, the levels of progesterone and testosterone metabolites are deeply affected in cerebrospinal fluid. These findings may have an important relevance in therapeutic and/or diagnostic field of multiple sclerosis.

Additional chromosomal abnormalities in Philadelphia-positive clone: adverse prognostic influence on frontline imatinib therapy: a GIMEMA Working Party on CML analysis.

Additional chromosomal abnormalities (ACAs) in Philadelphia-positive cells have been reported in ∼ 5% of patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP). Few studies addressing the prognostic significance of baseline ACAs in patients treated with imatinib have been published previously. The European LeukemiaNet recommendations suggest that the presence of ACAs at diagnosis is a "warning" for patients in early CP, but there is not much information about their outcome after therapy with tyrosine kinase inhibitors. To investigate the role of ACAs in early CP CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective trials of the Gruppo Italiano Malattie Ematologiche dell'Adulto Working Party on CML: 378 patients were evaluable and ACAs occurred in 21 patients (5.6%). The overall cytogenetic and molecular response rates were significantly lower and the time to response was significantly longer in patients with ACAs. The long-term outcome of patients with ACAs was inferior, but the differences were not significant. The prognostic significance of each specific cytogenetic abnormality was not assessable. Therefore, we confirm that ACAs constitute an adverse prognostic factor in CML patients treated with imatinib as frontline therapy.

Third trimester amniotic fluid cells with the capacity to develop neural phenotypes and with heterogeneity among sub-populations.

PURPOSE: Our aim was the search for new sources of cells potentially useful for central nervous system regenerative medicine. Extra-embryonic tissues are promising sources of pluripotent stem cells. Among these, human second-trimester amniotic fluid (AF) contains cell populations exhibiting self-renewal capacity, multipotency and the expression of embryonic cell markers. METHODS: Here we report the properties of the easily available third-trimester AF cells (AFCs). Different cell types from 6 of 9 AF samples were separated, expanded, and characterized by assessing their morphological, proliferative, and differentiative properties. RESULTS: All isolated cultures presented CD105, CD90 and CD73 mesenchymal markers, whereas they differed among themselves in CD117, CD146, CD31, NG2 and CD133 expression. Their doubling time and telomere length were conserved throughout many passages. Importantly, immunofluorescence and Real-time PCR showed that, during their proliferative state and differentiation, several cultures expressed neuronal and glial markers such as nestin, GFAP, ß-tubulin III and neurofilament H indicating their potential attitude towards a neural fate. Indeed, these cells showed a rather poor capacity to differentiate in adipogenic and osteogenic lineages. CONCLUSIONS: In this work we report that cells with neural differentiation capability can be isolated from third-trimester AF, such properties could be useful for neuro-regenerative purposes.

Chromosome abnormalities additional to the Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications.

Additional chromosome abnormalities (ACAs) occur in less than 10% of cases at diagnosis of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). In some cases, on the basis of the persistence of the ACAs in Ph-negative cells after response to imatinib, a secondary origin of the Ph chromosome has been demonstrated. In this study, the possible prognostic value of this phenomenon was evaluated. Thirty-six Ph-positive CML patients were included in the study. In six patients, ACAs persisted after the disappearance of the Ph. A complete cytogenetic response (CCR) was obtained in five of these six patients, and five of six also had a high Sokal score. In all the other cases, ACAs disappeared together (in cases of response to therapy with imatinib) or persisted with the Ph (in cases of no response to imatinib). In the former cases, the primary origin of the Ph was demonstrated. CCR was obtained in 22 cases (17 with low to intermediate Sokal scores), while no response was observed in 8 patients (5 with a high Sokal score). Sokal score seems to maintain its prognostic value for patients in whom the Ph occurs as a primary event, but not in those in whom it occurs as a secondary one.

Cytogenetic characterization and cell cycle analysis of three human colon adenocarcinoma cell lines: comparison between two- and three-dimensional cell culture systems.

The aim of the study was to investigate whether changes in the pattern of gene copy number and cell cycle were present passing from the two- to the three-dimensional cell culture system. We used three human colon adenocarcinoma cell lines grown two- and three-dimensionally. We analyzed morphology, karyotype, chromosomal gain and losses, and cell cycle. In three-dimensional cell cultures the growth is delayed and arrested in G1 phase without specific rearrangements in the three-dimensional cultures compared to the two-dimensional cultures. These data suggest that the differences between the two- and three-dimensional cell culture systems do not involve chromosomal rearrangements.

Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

De novo balanced chromosome rearrangements in prenatal diagnosis.

OBJECTIVE: We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome. METHOD: By means of a questionnaire, data on 269.371 analyses performed from 1983 to 2006 on amniotic fluid, chorionic villus and fetal blood samples were collected. RESULTS: A total of 246 balanced anomalies were detected at frequencies of 72% for reciprocal translocations, 18% for Robertsonian translocations, 7% for inversions and 3% for complex chromosome rearrangements. The total frequencies of balanced rearrangements were 0.09%, 0.08% and 0.05% on amniotic fluid, chorionic villus and fetal blood samples. CONCLUSION: A preferential involvement of chromosomes 22, 7, 21, 3, 9 and 11 and a less involvement of chromosomes X, 19, 12, 6 and 1 was observed. A nonrandom distribution of the breakpoints across chromosomes was noticed. Association in the location of recurrent breakpoints and fragile sites was observed for chromosomes 11, 7, 10 and 22, while it was not recorded for chromosome 3. The rate of pregnancy termination was about 20%, with frequencies decreasing from complex chromosomal rearrangements (33%), reciprocal translocations (24%) to inversions (11%) and Robertsonian translocations (3%).

Resveratrol inhibits cell growth in a human cholangiocarcinoma cell line.

BACKGROUND/AIMS: Cholangiocarcinoma is a devastating tumour with a poor prognosis. An efficient therapy is unavailable in unoperable patients and new drugs are widely sought for and required. Resveratrol (RES) is a natural molecule with a reported anticancer effect, evaluated on different tumour cell lines. We tested the efficacy of RES on a cholangiocarcinoma cell line for the first time. METHODS: We used the human SK-ChA-1 cell line, cultured in the classical two-dimensional model and in the three-dimensional spheroids. After RES exposure morphology, cell viability (colony-forming assay), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and cancer antigen (CA) 19-9 medium releases, cellular transglutaminase activity, karyotype and cell cycle were evaluated. RESULTS: Resveratrol inhibited cell growth in both the cell culture systems used (from -15 to -80% vs untreated controls) and induced a 40-fold increase of LDH and ALP activities in the culture medium. Also, transglutaminase (TG) activity increased in the cell lysates, together with a cell cycle perturbation characterised by an accumulation in the G(1)/S phase. Karyotype and CA 19-9 expression were not influenced by the treatment. CONCLUSIONS: The observed cytotoxic effect of RES on the human cholangiocarcinoma SK-ChA-1 cell line cultured two- and three-dimensionally suggests to further analyse its chemotherapic/chemopreventive possibilities for this kind of cancer.

Survivin expression in "low-risk" and "high-risk" myelodysplastic syndromes.

Apoptosis has a crucial role in myelodysplastic syndromes (MDS), being responsible of the ineffective hematopoiesis characteristic of the disease. Apoptosis rate is elevated in "early phase" MDS, whereas it diminishes during disease progression to acute leukemia, consensually to the acquisition of independent growth features. Survivin is a member of the inhibitor of the apoptosis (IAP) family, with the bifunctional role of suppressing apoptosis while facilitating cell cycle progression. We investigated Survivin mRNA levels by real-time quantitative reverse transcriptase PCR analysis and Survivin protein expression by immunohistochemistry in 49 bone marrow (BM) aspirates and in 17 BM biopsies (BMB) from MDS patients. Survivin mRNA levels were higher in MDS than in control group (1.68 +/- 1.46 vs 0.25 +/- 0.22; p < 0.0001). MDS patients with low or INT1 International Scoring System for Evaluating Prognosis (IPSS) displayed higher levels of Survivin mRNA in comparison to INT2 or high IPSS (1.91 +/- 1.51 vs 0.88 +/- 0.95; p = 0.0058). Survivin protein immunoreactivity was evaluated as Survivin index S ((i)) and calculated according to the formula: S ((i)) = % of Survivin positive cells x BMB cellularity / 100. Survivin index was higher in the MDS group than in normal BM (p = 0.05). Moreover, in eight cases in which BM aspirates and trephine biopsy were available, we found a significant association between the level of Survivin mRNA and protein expression (p = 0.011). In conclusion, this study demonstrates increased levels of Survivin in MDS compared to normal controls. Moreover, higher levels of transcripts are related to "low-risk" MDS. Our results suggest an active role of Survivin in normal and in myelodysplastic hematopoiesis.