sFlt-1 and CA 15.3 are indicators of endothelial damage and pulmonary fibrosis in SARS-CoV-2 infection.

COVID-19 pandemic led to a worldwide increase of hospitalizations for interstitial pneumonia with thrombosis complications, endothelial injury and multiorgan disease. Common CT findings include lung bilateral infiltrates, bilateral ground-glass opacities and/or consolidation whilst no current laboratory parameter consents rapidly evaluation of COVID-19 risk and disease severity. In the present work we investigated the association of sFLT-1 and CA 15.3 with endothelial damage and pulmonary fibrosis. Serum sFlt-1 has been associated with endothelial injury and sepsis severity, CA 15.3 seems an alternative marker for KL-6 for fibrotic lung diseases and pulmonary interstitial damage. We analysed 262 SARS-CoV-2 patients with differing levels of clinical severity; we found an association of serum sFlt-1 (ROC AUC 0.902, decision threshold > 90.3 pg/mL, p < 0.001 Sens. 83.9% and Spec. 86.7%) with presence, extent and severity of the disease. Moreover, CA 15.3 appeared significantly increased in COVID-19 severe lung fibrosis (ICU vs NON-ICU patients 42.6 ± 3.3 vs 25.7 ± 1.5 U/mL, p < 0.0001) and was associated with lung damage severity grade (ROC AUC 0.958, decision threshold > 24.8 U/mL, p < 0.0001, Sens. 88.4% and Spec. 91.8%). In conclusion, serum levels of sFlt-1 and CA 15.3 appeared useful tools for categorizing COVID-19 clinical stage and may represent a valid aid for clinicians to better personalise treatment.

Human Leukocyte Antigen-DR Isotype Expression in Monocytes and T Cells Interferon-Gamma Release Assay in Septic Patients and Correlation With Clinical Outcome.

BACKGROUND: Sepsis is a life-threatening dysregulated host response to infection responsible of multiple organs dysfunction (Sepsis-3 International Consensus Definition), during which clinical outcome is a balance between inflammation and immune suppression. Monocytes and lymphocytes may play an important role in immune paralysis, and their impaired functional activity can decrease overall immune system efficiency. We evaluated sepsis-induced changes in monocytes human leukocyte antigen-DR isotype (HLA-DR) expression and T cell capacity of interferon (IFN)-γ production in relation with patient's clinical outcome. METHODS: Analysis of HLA-DR expression on blood monocytes (mHLA-DR) was performed in 55 patients with high procalcitonin (hPCT, > 0.5 ng/mL,) and suspected/confirmed sepsis, and 20 controls. HLA-DR absolute quantification and IFN-γ release assay were monitored in 16 septic patients for 4 weeks following sepsis confirmation. RESULTS: Cytofluorimetric analysis revealed a significant decrease of mHLA-DR percentage in septic patients with adverse outcome compared to patients with better clinical outcome (88.4% vs. 98.6% with P < 0.05), in combination with a significant decrease of absolute number of HLA-DR molecules per monocyte (P < 0.05, starting at 1 week of follow-up). Lymphocytes stimulation with phytohemagglutinin (PHA), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) showed a severe declining of IFN-γ release related to fatal clinical outcome of patients. CONCLUSIONS: This immunologic anergy of innate and adaptative immunity showed an early immune paralysis during sepsis which appears correlated with the impairment of clinical outcome.

IgM and IgG Profiles Reveal Peculiar Features of Humoral Immunity Response to SARS-CoV-2 Infection.

The emergence of coronavirus disease 2019 (COVID-19) is globally a major healthcare threat. There is little information regarding the mechanisms and roles of the humoral response in SARS-CoV-2 infection. The aim of this study was to analyze the antibody levels (IgM and IgG) by chemiluminescence immunoassay in 54 subjects positive to SARS-CoV-2 swab test in relation to their clinical status (whether asymptomatic, pauci-symptomatic or with mild, sever or critical symptoms), the time from the symptom onset, sex, age, and comorbidities. Overall, the presence of comorbidities and the age of subjects were associated with their clinical status. The IgG concentrations were significantly higher in patients who developed critical and severe symptoms and seemed to be independent from age, sex and comorbidities. IgG titers peaked around day 60, and then began gradually to drop, decreasing by approximately 50% on the 180th day, while the IgM titers progressively decreased as early as the tenth day, but they could be detected even at later time points. Despite the small number of individuals, some peculiar characteristics of the humoral response in COVID-19 emerged. We observed a high inter-individual variability, an ephemeral IgG half-life in several patients, and a persistence of IgM in others.

Effects of Influenza Vaccination on the Response to BNT162b2 Messenger RNA COVID-19 Vaccine in Healthcare Workers.

BACKGROUND: Vaccine-induced immunity is at present the main strategy to stop the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent evidences suggested a protective effect of influenza vaccination against coronavirus disease 2019 (COVID-19) severity, while impact on the immune response to BNT162b2 messenger RNA (mRNA) vaccine is under investigation. METHODS: We aimed to evaluate this aspect in a cohort of 297 healthcare workers (108 males, 189 females) after seasonal influenza vaccination compared to no-flu-vaccination. VAX+ (165 individuals; 63 males and 102 females) had tetravalent influenza vaccine, and VAX- (132 individuals; 45 males and 87 females) had no flu vaccination. Anti-spike-receptor binding domain (RBD) level was tested 15 - 70 days after BNT162b2 second inoculum. RESULTS: Increased antibody response was observed in total VAX+ compared to VAX- (2,047.4 vs. 1,494.2 binding antibody unit (BAU)/mL, P = 0.0039), independently from gender and body mass index (BMI). Younger total individuals (< 35 years) showed significant increase of the level of binding antibodies (2,184.8 vs. 1,590.9 BAU/mL, P = 0.0038) compared to ≥ 35 years; young/old difference was lost restricting to VAX+ subgroup. Flu vaccinations appear associated to better antibody response in older individuals (P = 0.027, ≥ 35 years VAX+ vs. VAX-). A decreasing trend during time was observed for both VAX+ and VAX-, except for < 35 years VAX- individuals. Early response was higher in VAX+ compared to VAX-; however a more rapid waning was observed in VAX+ subjects. CONCLUSIONS: Our data showed better antibody response to SARS-CoV-2 vaccine in subjects already vaccinated against seasonal influenza; this may represent one of the mechanisms underlying the cross-protective effects of influenza vaccination against heterologous infections reported in recent epidemiological studies.

Flow Cytometric Analysis of Monocytes Polarization and Reprogramming From Inflammatory to Immunosuppressive Phase During Sepsis.

Sepsis outcome is determined by a balance between inflammation and immune suppression. We aimed to evaluate monocytes polarization and reprogramming during these processes. We analyzed 93 patients with procalcitonin level >0.5 ng/mL (hPCT) and suspected/confirmed sepsis, and 84 controls by analysis of CD14, CD16 and HLA-DR expression on blood monocytes using fluorescent labeled monoclonal antibodies and BD FACS CANTO II. Complete blood cell count, procalcitonin and other biochemical markers were evaluated. Intermediate monocytes CD14++CD16+ increased in hPCT patients (including both positive and negative culture) compared to controls (13.6% ± 0.8 vs 6.2% ± 0.3, p<0.001), while classical monocytes CD14++CD16-were significantly reduced (72.5% ± 1.6 vs 82.6% ± 0.7, p<0.001). Among hPCT patients having positive microbial culture, the percentage of intermediate monocytes was significantly higher in septic compared with non-septic/localized-infection patients (17.4% vs 11.5%; p<0.05) whilst the percentage of classical monocytes was lower (68.0% vs 74.5%). Three-four days following the diagnosis of sepsis, HLA-DR expression on monocyte (mHLA-DR) was lower (94.3%) compared to controls (99.4%) (p<0.05). Septic patients with the worst clinical conditions showed higher incidence of secondary infections, longtime hospitalization and lower HLA-DR+ monocytes compared to septic patients with better clinical outcome (88.4% vs 98.6%, p=0.05). The dynamic nature of sepsis correlates with monocytes functional polarization and reprogramming from a pro-inflammatory CD14++CD16+ phenotype in non-septic hPCT patients to a decrease of HLA-DR surface expression in hPCT patients with confirmed sepsis, making HLA-DR reduction a marker of immune-paralysis and sepsis outcome. Analysis of monocytes plasticity opens to new mechanisms responsible for pro/anti-inflammatory responses during sepsis, and new immunotherapies.

Soluble Fms-Like Tyrosine Kinase-1 Is A Marker of Endothelial Dysfunction During Sepsis.

BACKGROUND: Sepsis is currently defined as a life-threatening organ dysfunction caused by a deregulated host response to infection. There is increasing evidence that the endothelium plays a crucial and pathogenic role in sepsis. Profound alterations of the endothelium associated with sepsis include increased leucocytes adhesions, shift to a procoagulant state, vasodilatation, altered barrier function with more permeable capillaries and tissue edema. The vascular endothelial growth factor (VEGF) pathway is involved in the control of microvascular permeability and has been involved in the pathogenesis of conditions associated with endothelial barrier disruption such as sepsis. sFlt-1 is a soluble variant of the VEGF receptor (Fms-like tyrosine kinase-1, Flt-1 or VEGFR-1) able to down-regulate the effects of VEGF by decreasing its signaling. We investigated the possible involvement of sFlt-1 as biomarker of endothelial alteration during sepsis, organ dysfunction and death. METHODS: Serum levels of s-Flt1 were measured in 170 hospitalized patients (77 with sepsis, confirmed by positive blood culture), and in 18 healthy volunteers. The sequential organ failure assessment (SOFA) score was determined by using biochemical and clinical parameters. In a small number of patients (9 individuals), s-Flt1 concentration was evaluated after negativization of the blood culture. RESULTS: Serum level of s-Flt1 was significantly higher in septic patients than blood culture-negative patients (277.7 ± 52.7 and 133.4 ± 12.4 pg/mL, respectively, P = 0.0088), both groups of patients had significantly higher concentration of sFlt-1 than healthy individuals (78.9 ± 2.5 pg/mL). Among sepsis cases, 68% was caused by Gram-negative bacteria, 27% by Gram-positive bacteria and 8% by Candida species. Serum level of s-Flt1 showed a significant difference between Gram-negative (274.1 pg/mL) and Gram-positive (145.7 pg/mL) sepsis. SOFA score (evaluated in 20 patients with sFlt-1 >190 pg/mL) showed a positive trend of correlation with the increasing sFlt-1 level. After blood culture negativization, serum level of sFlt-1 decreased (37%). CONCLUSION: Our findings confirm, in a larger population of patients with sepsis, recent evidences that sFlt-1 levels are higher in patients with complicated-sepsis that evolve to septic shock and suggest that sFlt-1 could be a useful biomarker for sepsis severity. An anti-VEGF effect mediated by sFlt-1 could be hypothesized as salvage compensatory mechanism activated in response to sepsis.

Seropositivity for West Nile Virus Antibodies in Patients Affected by Myasthenia Gravis.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles. Specific auto-antibodies against acetylcholine receptor (AChR) are present in the majority of MG patients, although the main cause behind its development still remains unclear. Recently MG development following West Nile virus (WNV) infection has been described in patients without any earlier evidence of MG. It is known that infectious agents trigger immune response and occasionally initiate autoimmune disease. WNV, the causative agent of both benign illness and neuroinvasive disease, has become endemic in many countries in all continents. METHODS: In the present study, 29 patients (15 males and 14 females, 19 - 78 years old) with confirmed diagnosis of MG and elevated levels of AChR autoantibodies were screened for the presence of serum anti-WNV antibodies and compared to a similar population affected by different autoimmune diseases. Indirect immunofluorescent antibody technique was used to evaluate the reaction of patients' sera on cells infected by WNV. RESULTS: Positive fluorescent signals for anti-WNV IgG were obtained in 17% of MG patients, although no clinical manifestations related to WNV infection were reported. These results are in agreement with previous data and appear of great interest in the understanding of the pathogenic autoimmune mechanisms at the bases of MG development. CONCLUSION: As already observed in other human autoimmune diseases, pathogen-triggered autoimmunity could be involved in MG by breaking immunological self-tolerance through possible mechanisms of molecular mimicry between virus proteins and AChR subunits. In predisposed individuals, WNV infection could also represent an additional risk factor to initiate MG.

Selective genetic analysis of myoma pseudocapsule and potential biological impact on uterine fibroid medical therapy.

OBJECTIVE: Mutations in Mediator Complex Subunit 12 (MED12) gene are typical genomic aberrations, commonly detected in a high percentage of uterine leiomyomas (ULs). The aim of this investigation was to define the fibroid or non-tumor origin of uterine leiomyoma pseudocapsule (PC) surrounding fibroids and its possible therapeutic targets in uterine fibroid management. RESEARCH DESIGN AND METHODS: A non-randomized observational study was performed on 36 women, not subjected to any previous drug treatment, undergoing laparoscopic intracapsular myomectomy. Specimens of myometrium (UM), ULs and corresponding PCs were sampled to analyze MED12 gene status, by direct sequencing of exon 2. MAIN OUTCOME MEASURES: Defining the status of MED12 gene in PCs associated to ULs harboring mutations. RESULTS: PCs always showed a wild type MED12 gene status, even when associated to a UL harboring a specific MED12 aberration. CONCLUSIONS: The wild-type status of MED12 gene in the PCs indicates the non-tumoral origin of this structure: it appears as a protective structure for the healthy tissue that could enhance regenerative mechanisms. The limitations of this study, as the restrained number of patients, will be solved in the future extending the analysis to a larger cohort of women, as tester of such pharmacological treatments on PC.

The opioid neuropeptides in uterine fibroid pseudocapsules: a putative association with cervical integrity in human reproduction.

The myoma pseudocapsule (MP) is a fibro-vascular network rich of neurotransmitters, as a neurovascular bundle, surrounding fibroid and separating myoma from myometrium. We investigated the distribution of the opioid neuropeptides, as enkephalin (ENK) and oxytocin (OXT), in the nerve fibers within MP and their possible influence in human reproduction in 57 women. An histological and immunofluorescent staining of OXT and ENK was performed on nerve fibers of MP samples from the fundus, corpus and isthmian-cervical regions, with a successive morphometric quantification of OXT and ENK. None of the nerve fibers in the uterine fundus and corpus MPs contained ENK and the nerve fibers in the isthmian-cervical region demonstrated an ENK value of up to 94 ± 0.7 CU. A comparatively lower number of OXT-positive nerve fibers were found in the fundal MP (6.3 ± 0.8 CU). OXT-positive nerve fibers with OXT were marginally increased in corporal MP (15.0 ± 1.4 CU) and were substantially higher in the isthmian-cervical region MP (72.1 ± 5.1 CU) (p < 0.01). The distribution of OXY neurofibers showed a slight into the uterine corpus, while are highly present into the cervico-isthmic area, with influence on reproductive system and sexual disorders manifesting after surgical procedures on the cervix.

A new germline stop codon mutation in exon 15 of the APC gene predisposing to familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder related to germline mutations of the adenomatous polyposis coli (APC) gene. It is characterized by the detection of numerous adenomatous polyps that, if untreated, develop into colorectal cancer. We studied an Italian family with FAP history and the related colorectal tumor sample of the proband. Sequencing analysis of blood samples revealed the presence of a never-reported germline mutation in the APC gene (exon 15): an heterozygous G deletion at position c.2126 resulting in a premature stop codon (p.Gly721GlufsX6) and in a truncated protein. This mutation was also identified in the colorectal tumor tissue, together with a second known pathogenic heterozygotic somatic mutation, c.4348C>T (p.Arg1450X), which generates a premature truncated protein. The novel identified germline mutation is therefore related to FAP and, in accordance with Knudson's "two hit" hypothesis, can be considered the first event predisposing to the insurgence of colorectal cancer in these patients. The somatic hit inactivating the second allele of the APC gene is located in the mutation cluster region of the gene; this is not a random event since it depends on the position of the germline mutation. The inactivation of APC generates the neoplastic growth advantage to the cell.

Small-Sized Clone of T Cells in Multiple Myeloma Patient after Auto-SCT: T-LGL Leukemia Type or Clonal T-Cell Aberration?

Second cancers and particularly postransplant lymphoproliferative disorders (PTLDs) are extremely rare in patients undergoing autologous peripheral blood stem cell transplantation (auto-SCT). We report the case of clonally rearranged T-cell expansion which occurred after auto-SCT for Multiple Myeloma (MM). Does asymptomatic clonal T-cell large granular lymphocytic proliferation, in our experience, represent either a secondary cancer after auto-SCT or clonal T cell aberration or derive from expansion of coexisting undetected small-sized clone of T cells?

The mRNA-HPV test utilization in the follow up of HPV related cervical lesions.

UNLABELLED: The biomolecular follow up of Human Papilloma Virus (HPV) is widely investigated in patients treated for HPV related cervical lesions, since the HPV-mRNA test is more specific and have a higher positive predictive value for CIN2-3 in triage of high risk (HR) women and in follow-up of women treated for CIN2/3. MATERIAL & METHODS: We investigated, during a 5 years' study, a cohort of patients divided in: group 1, patients at high risk for HPV-infections, and group 2, women diagnosed for CIN2/3, Cervicocarcinoma in situ (CIS) and Adenocarcinoma in situ (AIS) and surgically treated. The overall scheduled follow up was repeated each 6 months by: Pap Test, HPV-DNA test, m-RNA-HPV test and, in case of CIN2/3, CIS and AIS, also by colposcopy and biopsy. RESULTS: The follow up involved a total of 203 women: 85 women with mRNA-HPV positive test and 118 patients surgically treated for CIN2/3, CIS and AIS. In the group 1, the long term follow up detected, after one year, 32 positive mRNA-HR HPV women and, of these, after more than 2 years, 37.5% developed CIN1 and 21.8% developed CIN2/3. Similarly, in the follow up of group two, women with abnormal Pap test showed positivity of mRNA HR-HPV in 71.4% of cases even after 6 months; 65% of these developed a CIN1 within 2.5 years and 20% had CIN2/3 after 2.3 years. CONCLUSIONS: Study results indicate either that patients with mRNA HR-HPV positive controls, on average, after 12 months are all at risk of progression to CIN1 and CIN2/3, or the higher specificity of mRNA-HPV test than Pap Test in follow up of surgical treated patients. This investigation confirmed a strong association between HR mRNA-HPV presence and risk of neoplastic progression.

Prevalence and distribution of human papillomavirus genotype in south eastern Italy, in the period 2006-2011: implications for intervention.

Persistent infection of High Risk (HR) Human papillomavirus (HPV) infection can lead to cervical cancer. The HPV genotypes are found worldwide, but important regional variations have been found. For a population-based HPV type prevalence study to assess the effect of existing and new prevention methods, frequently updated information on the burden of cervical cancer is essential. We evaluated the prevalence of HPV genotypes in a volunteer population screened for cervical cancer at the Local Health Unit (LHU) of Lecce. A total of 9,720 women were studied. The tests were performed by INNO-Lipa HPV Genotyping and LINEAR ARRAY HPV Genotyping Test. The overall HPV prevalence was 29.7% (95% CI, 28.8-30.6) for any HPV DNA. The prevalent type for all age groups was HPV 16 (7.4%; CI, 6.9-7.9) followed by HPV 31 (3.4%; CI, 3.0-3.7), 51 (3.0%; CI, 2.6-3.3), 52 (2.7%; CI, 2.3-3.0) and 58 (2.4%; CI, 2.1-2.7). HPV 53 was the most common low-risk HPV type with prevalence rate of 3.5 (CI, 3.1-3.8), followed by HPV 66 (3.0; CI, 2.6-3.3), 6 (2.9; CI, 2.6-3.2) and 42 (2.5; CI, 2.2-2.8). Multiple infections were present in 13.6% of HPV-tested women (CI, 12.9-14.3). Among these, the most common combination was of HPV 16 and HPV 52 genotypes. This study reports high prevalence of HPV infection and may serve as a valuable reference for assessing the impact of HPV vaccination programs. Furthermore, it supports the need for new vaccines that contain the most common HPV genotypes present in the population.

NT, NPY and PGP 9.5 presence in myomeytrium and in fibroid pseudocapsule and their possible impact on muscular physiology.

The uterine myoma pseudocapsule is a neurovascular bundle surrounding fibroid, containing neuropeptides, probably involved in uterine scar healing. We studied neurotensin (NT), neuropeptide tyrosine (NPY), and protein gene product 9.5 (PGP 9.5) nerve fibres in the pseudocapsule neurovascular bundle of intramural uterine fibroids on 67 no pregnant women by intracapsular myomectomy sparing the neurovascular bundle, sampling full thickness specimens of the pseudocapsule of uterine fibroids (PUF) and normal myometrium (NM) obtained from the fundus uteri (FU) and the uterine body (UB). The samples were sent for histological and immunofluorescent analyses and compared by morphometrical quantification. The Conventional Unit (C.U.) difference of NT, NPY, and PGP 9.5 nerve fibres was statistically analyzed. Our results showed that NT, NPY, and PGP 9.5 neurofibers are almost equally present in PUF as in NM of a no pregnant uterus. As all of these neuropeptides are present in the uterine muscle and can affect muscle contractility, uterine peristalsis and muscular healing. A myomectomy respecting the pseudocapsule neurofibers should facilitate smooth muscle scarring and promote restoration of normal uterine peristalsis with a possible positive influence on fertility.

Serum proteomic profile of cutaneous malignant melanoma and relation to cancer progression: association to tumor derived alpha-N-acetylgalactosaminidase activity.

Currently clinical outcome in melanoma is not predictable by known serum biomarkers. The only reliable tool for the diagnosis of this tumor is the histopathological assay after surgical removing. We used a proteomic approach in order to identify novel non-invasive serum biomarkers of melanoma. Serum proteomic maps showed different patterns in relation to the presence and progression of the tumor in five regions of the map. Differently expressed spots were identified by MALDI-TOF mass spectrometry. Significant increases of expression were found for transthyretin (TTR) and angiotensinogen (AGT) while vitamin D binding protein (DBP) expression was decreased in presence of melanoma. Interestingly, protein expression came back to control values in stages I and II of the disease after 1 month since surgical removal of suspected melanoma. We related the decrease of DBP spot to the impaired immune response of cancer patients. In fact cancer cells release the alpha-N-acetylgalactosaminidase that can deglycosylate DBP thus interfering with the immune cascade response in which DBP is involved, leading to immunosuppression in melanoma patients. Specific enzymatic activity of serum alpha-N-acetylgalactosaminidase was significantly increased in stage III melanoma patients, but not in early stages. This enzymatic assay may provide a non-invasive way of evaluation of melanoma severity.

A proteomic approach for the characterization of C677T mutation of the human gene methylenetetrahydrofolate reductase.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate (CH2H4folate) to methyltetrahydrofolate (CH3H4folate). The C677T mutation is a common polymorphism of the human enzyme that leads to the replacement of Ala222Val, thermolability of MTHFR, and mild elevation of plasma homocysteine levels. A mild hyperhomocysteinemia is known to be risk factor for cardiovascular and thrombotic diseases, ischemic stroke, neural tube defects, late on-set dementia, and pregnancy complications. Human plasma of subjects carrying the C677T mutation in the MTHFR gene has been investigated for their protein pattern in order to identify novel molecular hallmarks. 2-D analysis of the plasma protein allowed the identification of a specific pattern associated with the TT mutant genotype. Noteworthy, we found one spot shifted to a more basic pI in mutant individuals, and MS identification corresponded to vitamin D-binding protein (DBP or group component (Gc) globulin). MS/MS peptide sequencing allowed to discriminate different allelic variants in the investigated clinical groups. These data confirmed by molecular genetic analysis highlight the novel association between the C677T MTHFR genotype with the Gc2 polymorphism of the DBP. Moreover, we found a quantitative reduction of Apolipoprotein A-I in mutant individuals, which was associated, in previous studies by others to an increased cardiovascular risk.

Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts.

An extensive analysis has been carried out of mitochondrial biochemical and bioenergetic properties of fibroblasts, mostly skin-derived, from a large group of subjects ranging in age between 20 wk fetal and 103 yr. A striking age-related change observed in a fundamental process underlying mitochondrial biogenesis and function was the very significant decrease in rate of mitochondrial protein synthesis in individuals above 40 yr. The analysis of endogenous respiration rate revealed a significant decrease in the age range from 40 to 90 yr and a tendency to uncoupling in the samples from subjects above 60 yr. A surprising finding was the occurrence of a subgroup of individuals >or=90 yr old whose skin fibroblasts exhibited an exceptionally high respiration rate. This high rate was not due to respiration uncoupling, rather pointing to a compensatory phenomenon, not involving an increase in mtDNA content, in the corresponding skin fibroblast populations, or, possibly, to a selection of a different cell type secondary to more extensive dermal atrophy. The most important aging-related phenotypic effects observed were those that affected the cell oxidative phosphorylation (OX-PHOS) capacity. These were, in particular, the very significant reduction in the ratio of uncoupled to oligomycin-inhibited endogenous respiration observed in intact fibroblasts, which pointed to a decrease with donor's age in the control of respiration by the mitochondrial membrane potential, the very significant decrease in efficiency of OX-PHOS, as determined by novel in situ measurements of P:O ratios, and, consistent with these results, the very significant reduction in the respiratory control ratios. These findings clearly pointed to a dramatic mitochondrial dysfunction, which would lead to a decrease in ATP synthesis rate, with the observed decline in mitochondrial protein synthesis rate being a likely contributing factor. These observations have important implications for understanding the biology of aging, as well as the pathogenesis of aging-related degenerative diseases.

A novel mutation in the SURF1 gene in a child with Leigh disease, peripheral neuropathy, and cytochrome-c oxidase deficiency.

We report a 16-month-old boy with psychomotor regression, muscle hypotonia, peripheral neuropathy, and lactic acidosis. Brain magnetic resonance imaging showed a bilateral abnormal signal in the substantia nigra and in the subthalamic nucleus, suggestive of Leigh disease. Histochemical analysis of skeletal muscle showed decreased cytochrome-c oxidase activity. Biochemical analysis of respiratory chain enzymes in muscle homogenate and in cultured fibroblasts showed isolated cytochrome-c oxidase deficiency. Western blot analysis in fibroblasts showed the absence of Surf1 protein. Genetic analysis of the SURF1 gene revealed that the patient was compound heterozygous for a previously reported mutation at the splice-junction site of intron 3 (240 + 1G > T), and for a novel 4-bp deletion in exon 6 (531_534delAAAT). Our data further enlarge the spectrum of mutations in SURF1 gene in patients with Leigh disease and cytochrome-c oxidase deficiency, contributing to better characterization of the clinical and neuroradiologic features of this group of patients for genotype-phenotype correlations.