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Test immunologici e virologici in persone con infezione da HIV a lungo termine senza progressione della malattia

Posted on Giugno 18, 2022Giugno 22, 2022 by Andrea

Large-scale automated hollow fiber bioreactor expansion of human umbilical cord-derived mesenchymal stromal cells for neurological disorders. Neurodegenerative diseases make up a large group of neurological diseases and remain one of the greatest challenges and burdens for humanity. Diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, stroke or spinal cord injury are often associated with astrogliosis (astrogliosis) with symptoms of inflammation.

  • The regenerative,  paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) can affect the above components, thus opening up new therapeutic possibilities for regenerative medicine.
  • Of particular interest are hMSCs derived from umbilical cord (UC) tissue due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard protocols for good manufacturing practice (   GMP   ) for the operational and large-scale isolation, characterization, expansion and comparison of the properties of UC-hMSC cells collected in T vials compared to use of a large-scale bioreactor system. .
  • Human UC-hMSC, isolated by culture technique from Wharton’s Jelly explant, was collected at 75% confluence and  cultured in tissue culture bottles. UC-hMSCs obtained before / after cryopreservation and after collection in the bioreactor were fully characterized in terms of immunomodulatory “mesenchymicity”, oncogenicity and genetic stability, aging and cell doubling properties and gene expression traits  . Our study demonstrates an efficient and simple technique for increasing UC-hMSC.
  • Collecting UC-hMSCs using classical and large-scale methods did not alter UC-hMSC aging, genetic stability or carcinogenicity in vitro. We observed comparable growth and immunomodulatory capacity of fresh, frozen and expanded UC-hMSC.
  • We found no differences in the ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages between classical and large-scale UC-hMSC expansion methods. Both methods made it possible to derive genetically stable cells with typical mesenchymal characteristics.
  • Interestingly, we found that UC-hMSC grown in a bioreactor showed significantly increased levels of neural growth factor (NGF) mRNA expression and reduced levels of insulin growth factor (IGF), while    IL4   , IL6, IL8,
  • TGFb and VEGF expression levels remained at a similar level. Cultivation of UC-hMSC using a large-scale automated closed bioreactor expansion system under  GMP conditions    does not alter basic “mesenchymal” characteristics and cell quality.
  • Our study was designed to pave the way for the translation of known basic research data on human UC-MSC into future clinical trials in patients with neurological and immunocompromised disorders.
  • Industrial production of UC-hMSC will then be approved by regulatory authorities under the criteria for advanced therapy medicinal products (ATMP) prior to clinical use and approval for use in patients.

The relaxation factor of endothelial origin produced and released by the arteries and veins is nitric oxide.

  1. The aim of this study was to determine whether nitric oxide (NO) is responsible for vascular smooth muscle relaxation induced by endothelial-derived relaxation factor (EDRF). EDRF is an unstable humoral substance released by arteries and veins that mediates the action of dependent endothelial vasodilators.
  2. NO is an unstable, endothelium-independent vasodilator that is released by vasodilators such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the effect of NO on vascular smooth muscle is very similar to that of EDRF. In the present study, the vascular effect of EDRF released from a pulmonary artery and a perfused vein of cattle was compared with that of NO delivered by overflow through endothelial strips of cascading arteries and veins.
  3. EDRF was indistinguishable from NO as both were unstable (t1 / 2 = 3-5 s), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced a comparable increase in the cyclic accumulation of   GMP    in the artery and vein, and this cyclic accumulation of   GMP    was inhibited by pyrogallol, oxyhemoglobin, potassium and methylene blue.
  4. EDRF was chemically identified as NO or a labile nitrous species by two procedures. First, like NO, EDRF released from newly isolated aortic endothelial cells reacted with hemoglobin to form nitrosylemoglobin.
  5. Second, EDRF and NO similarly promoted the diazotization of sulphanilic acid and produced the same reaction product after coupling with N- (1-naphthyl) ethylenediamine. Therefore, EDRF released from an artery and a vein has biological and chemical properties identical to NO.

The cyclic GMP  -AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

The presence of DNA in the cytoplasm of mammalian cells is a dangerous signal that triggers a host immune response, such as the production of type I interferons.   Cytosolic DNA induces interferons by producing cyclic guanosine-adenosine monophosphate monophosphate (cyclic    GMP  -AMP or cGAMP) which binds to the STING adapter protein and activates them.
Through biochemical fractionation and quantitative mass spectrometry, we identified cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. The overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner.
The impact of cGAS inhibited the activation of IRF3 and the induction of interferon-β by DNA transfection or DNA virus infection. cGAS binds to DNA in the cytoplasm and catalyzes the synthesis of cGAMP. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Providing epidemiological, virological and immunological parameters in HIV + patients defined as long-term progression (LTnP) may be the key to understanding the natural history of HIV disease and its therapeutic approach.

  1.  The aim of this study was to identify factors that delay the onset of HIV-related symptoms in HIV-infected patients with low progression.  We interviewed several people defined as LTnP according to the following criteria.
  2. Patients were asymptomatic with documented HIV infection for at least 8 years, had never been treated with antiretroviral therapy and had CD4 levels always above 500 / cmm. They were tested every 6 months for the following tests: virus isolation, viral strain characterization; Quantitative DNA-PCR; HIV p24 antigen in serum, plasma viraemia, neutralizing antibodies to autologous and heterologous primary isolates.

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Immunoassay involves a large panel of monoclonal antibodies to characterize lymphocyte phenotype, cell proliferation with mitogen and antigens (tetanus toxoid;   GMP    C. Albicans, p24 and HIV gp160), HIV-specific cytotoxicity (env and gag), and rate of spontaneous cell mortality. Lymphocyte culture supernatants (stimulated with PHA) are collected to measure  cytokines (IL2;    IL4   ; IL10; gamma IFN) and T cell clones grown to characterize cytokine phenotype and pattern. Here we provide only a summary of the immunological data collected.

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