Efficacy of COVID-19 mRNA vaccination in patients with autoimmune disorders: humoral and cellular immune response.

BACKGROUND: The impact of immunosuppressive therapies on the efficacy of vaccines to SARS-CoV-2 is not completely clarified. We analyzed humoral and T cell-mediated response after COVID-19 mRNA vaccine in immunosuppressed patients and patients with common variable immunodeficiency disease (CVID). PATIENTS: We enrolled 38 patients and 11 healthy sex- and age-matched controls (HC). Four patients were affected by CVID and 34 by chronic rheumatic diseases (RDs). All patients with RDs were treated by corticosteroid therapy and/or immunosuppressive treatment and/or biological drugs: 14 patients were treated with abatacept, 10 with rituximab, and 10 with tocilizumab. METHODS: Total antibody titer to SARS-CoV-2 spike protein was assessed by electrochemiluminescence immunoassay, CD4 and CD4-CD8 T cell-mediated immune response was analyzed by interferon-γ (IFN-γ) release assay, the production of IFN-γ-inducible (CXCL9 and CXCL10) and innate-immunity chemokines (MCP-1, CXCL8, and CCL5) by cytometric bead array after stimulation with different spike peptides. The expression of CD40L, CD137, IL-2, IFN-γ, and IL-17 on CD4 and CD8 T cells, evaluating their activation status, after SARS-CoV-2 spike peptides stimulation, was analyzed by intracellular flow cytometry staining. Cluster analysis identified cluster 1, namely the "high immunosuppression" cluster, and cluster 2, namely the "low immunosuppression" cluster. RESULTS: After the second dose of vaccine, only abatacept-treated patients, compared to HC, showed a reduced anti-spike antibody response (mean: 432 IU/ml ± 562 vs mean: 1479 IU/ml ± 1051: p = 0.0034), and an impaired T cell response, compared with HC. In particular, we found a significantly reduced release of IFN-γ from CD4 and CD4-CD8 stimulated T cells, compared with HC (p = 0.0016 and p = 0.0078, respectively), reduced production of CXCL10 and CXCL9 from stimulated CD4 (p = 0.0048 and p = 0.001) and CD4-CD8 T cells (p = 0.0079 and p = 0.0006). Multivariable General Linear Model analysis confirmed a relationship between abatacept exposure and impaired production of CXCL9, CXCL10, and IFN-γ from stimulated T cells. Cluster analysis confirms that cluster 1 (including abatacept and half of rituximab treated cases) showed a reduced IFN-γ response, as well as reduced monocyte-derived chemokines All groups of patients demonstrated the ability to generate specific CD4 T activated cells after spike proteins stimulation. After the third dose of vaccine, abatacept-treated patients acquired the ability to produce a strong antibody response, showing an anti-S titer significantly higher compared to that obtained after the second dose (p = 0.0047), and comparable with the anti-S titer of the other groups. CONCLUSIONS: Patients treated with abatacept showed an impaired humoral immune response to two doses of COVID-19 vaccine. The third vaccine dose has been demonstrated to be useful to induce a more robust antibody response to balance an impaired T cell-mediated one. All patients, exposed to different immunosuppressive drugs, were able to produce specific CD4-activated T cells, after spike proteins stimulation. TRIAL REGISTRATION: Local Ethical Committee NP4187.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Successful CAR-T cell therapy in a refractory MCL patient with bacterial, fungal and COVID-19 infection: a case report.

Background: The COVID-19 pandemic has had a significant impact on the management and care of onco-hematological patients, particularly those with lymphoproliferative disorders who are at higher risk for COVID-19 associated bacterial and fungal superinfections. Case presentation: We present the successful treatment of a 44-year-old male patient with refractory mantle cell lymphoma treated with chimeric antigen receptor T (CAR-T) cell therapy, despite concurrent COVID-19 infection. The patient developed grade II cytokine release syndrome, requiring admission to the intensive care unit. The CAR-T cells expanded effectively, and the patient achieved complete metabolic remission. During the treatment course, the patient experienced complications including COVID-19-associated pulmonary aspergillosis and a co-infection with Stenotrophomonas maltophilia and the SARS-CoV-2 omicron variant. Prompt antifungal and antibacterial therapy, along with appropriate COVID-19 treatment, led to the resolution of these infections. Dexamethasone was also administered to reduce inflammation and aid hematologic recovery. Despite the presence of multiple infections, the patient achieved complete remission of lymphoma, highlighting the effectiveness of CAR-T cell therapy in this high-risk patient. Conclusion: Despite the challenges posed by concurrent infections, the decision to proceed with CAR-T cell therapy in this patient proved to be successful, resulting in complete remission of lymphoma. Early initiation of supportive therapies and the use of dexamethasone contributed to the resolution of complications. This case underscores the importance of individualized decision-making and the potential benefits of CAR-T cell therapy in similar high-risk patients.

A community-acquired Legionnaires' disease outbreak caused by Legionella pneumophila serogroup 2: an uncommon event, Italy, August to October 2018.

In September 2018 in Brescia province, northern Italy, an outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 2 (Lp2) occurred. The 33 cases (two fatal) resided in seven municipalities along the Chiese river. All cases were negative by urinary antigen test (UAT) and most were diagnosed by real-time PCR and serology. In only three cases, respiratory sample cultures were positive, and Lp2 was identified and typed as sequence type (ST)1455. In another three cases, nested sequence-based typing was directly applied to respiratory samples, which provided allelic profiles highly similar to ST1455. An environmental investigation was undertaken immediately and water samples were collected from private homes, municipal water systems, cooling towers and the river. Overall, 533 environmental water samples were analysed and 34 were positive for Lp. Of these, only three samples, all collected from the Chiese river, were Lp2 ST1455. If and how the river water could have been aerosolised causing the LD cases remains unexplained. This outbreak, the first to our knowledge caused by Lp2, highlights the limits of UAT for LD diagnosis, underlining the importance of adopting multiple tests to ensure that serogroups other than serogroup 1, as well as other Legionella species, are identified.

B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation.

Combined antiretroviral therapy (cART) for HIV-1 dramatically slows disease progression among HIV+ individuals. Currently, lymphoma represents the main cause of death among HIV-1-infected patients. Detection of p17 variants (vp17s) endowed with B-cell clonogenic activity in HIV-1-seropositive patients with lymphoma suggests their possible role in lymphomagenesis. Here, we demonstrate that the clonogenic activity of vp17s is mediated by their binding to PAR1 and to PAR1-mediated EGFR transactivation through Gq protein. The entire vp17s-triggered clonogenic process is MMPs dependent. Moreover, phosphoproteomic and bioinformatic analysis highlighted the crucial role of EGFR/PI3K/Akt pathway in modulating several molecules promoting cancer progression, including RAC1, ABL1, p53, CDK1, NPM, Rb, PTP-1B, and STAT1. Finally, we show that a peptide (F1) corresponding to the vp17s functional epitope is sufficient to trigger the PAR1/EGFR/PI3K/Akt pathway and bind PAR1. Our findings suggest novel potential therapeutic targets to counteract vp17-driven lymphomagenesis in HIV+ patients.

Human Metapneumovirus Establishes Persistent Infection in Lung Microvascular Endothelial Cells and Primes a Th2-Skewed Immune Response.

Human Metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment.

Management of a Case of Peritonitis Due to Neisseria gonorrhoeae Infection Following Pelvic Inflammatory Disease (PID).

Pelvic inflammatory disease (PID), a serious infection in sexually active women, is one of the reasons for which females seek care in emergency departments and therefore represents an important public health problem. PID is the result of an endocervical infection with different microorganisms, which then ascend to the endometrium and fallopian tubes. Symptoms of PID may be mild and aspecific, making its diagnosis difficult. However, this clinical condition requires effective antibiotic treatment to reduce incidence of complications and late sequelae. We describe here a case of peritonitis as a complication of pelvic inflammatory disease (PID) due to Neisseria gonorrhoeae infection in a 49-year-old woman who presented at the Emergency Department with acute abdominal pain.

Role of Autophagy in Von Willebrand Factor Secretion by Endothelial Cells and in the In Vivo Thrombin-Antithrombin Complex Formation Promoted by the HIV-1 Matrix Protein p17.

Although the advent of combined antiretroviral therapy has substantially improved the survival of HIV-1-infected individuals, non-AIDS-related diseases are becoming increasingly prevalent in HIV-1-infected patients. Persistent abnormalities in coagulation appear to contribute to excess risk for a broad spectrum of non-AIDS defining complications. Alterations in coagulation biology in the context of HIV infection seem to be largely a consequence of a chronically inflammatory microenvironment leading to endothelial cell (EC) dysfunction. A possible direct role of HIV-1 proteins in sustaining EC dysfunction has been postulated but not yet investigated. The HIV-1 matrix protein p17 (p17) is secreted from HIV-1-infected cells and is known to sustain inflammatory processes by activating ECs. The aim of this study was to investigate the possibility that p17-driven stimulation of human ECs is associated with increased production of critical coagulation factors. Here we show the involvement of autophagy in the p17-induced accumulation and secretion of von Willebrand factor (vWF) by ECs. In vivo experiments confirmed the capability of p17 to exert a potent pro-coagulant activity soon after its intravenous administration.

The Synthetic Dipeptide Pidotimod Shows a Chemokine-Like Activity through CXC Chemokine Receptor 3 (CXCR3).

In recent years immunomodulators have gained a strong interest and represent nowadays an active expanding area of research for the control of microbial diseases and for their therapeutic potential in preventing, treating and reducing the morbidity and mortality of different diseases. Pidotimod (3-L-pyroglutamyl-L-thiaziolidine-4carboxylic acid, PDT) is a synthetic dipeptide, which possesses immunomodulatory properties and exerts a well-defined pharmacological activity against infections, but its real mechanism of action is still undefined. Here, we show that PDT is capable of activating tyrosine phosphorylation-based cell signaling in human primary monocytes and triggering rapid adhesion and chemotaxis. PDT-induced monocyte migration requires the activation of the PI3K/Akt signaling pathway and chemokine receptor CXCR3. Indeed, a mAb to CXCR3 and a specific receptor inhibitor suppressed significantly PDT-dependent chemotaxis, and CXCR3-silenced primary monocytes lost responsiveness to PDT chemoattraction. Moreover, our results highlighted that the PDT-induced migratory activity is sustained by the CXCR3A isoform, since CXCR3-transfected L1.2 cells acquired responsiveness to PDT stimulation. Finally, we show that PDT, as CXCR3 ligands, is also able to direct the migration of IL-2 activated T cells, which express the highest levels of CXCR3 among CXCR3-expressing cells. In conclusion, our study defines a chemokine-like activity for PDT through CXCR3A and points on the possible role that this synthetic dipeptide may play in leukocyte trafficking and function. Since recent studies have highlighted diverse therapeutic roles for molecules which activates CXCR3, our findings call for an exploration of using this dipeptide in different pathological processes.

Lymphomagenic properties of a HIV p17 variant derived from a splenic marginal zone lymphoma occurred in a HIV-infected patient.

Despite antiretroviral therapy, HIV+ individuals still have increased risk to develop lymphomas, including marginal zone lymphomas, suggesting that factors other than HIV-related immunosuppression are probably acting as lymphomagenic factors in the HIV setting. The possible pathogenic involvement of HIV p17 protein variants was investigated in a particularly informative case of HIV-related splenic marginal zone lymphoma, which was negative for oncogenic virus infections, thus allowing us to assess the possible direct contribution of these HIV-encoded proteins to lymphomagenesis. The presence of p17 protein was analyzed by immunohistochemistry in lymphoma tissue. Recombinant p17 protein derived from the dominant sequence detected in plasma and lymphoma biopsy was characterized for B-cell proliferation, clonogenicity in soft agar, in vitro tube formation and wound healing. Intracellular signaling was investigated by immunoblotting. HIV p17 protein was detected in reactive lymphoid follicles but not within lymphoma cells. An identical dominant variant p17 sequence, p17-Lyrm, carrying a 117 to 118 Ala-Ala insertion was detected in both plasma and lymphoma tissue. Recombinant p17-Lyrm enhanced B-cell proliferation and clonogenicity promoted the formation of capillary-like structures and enhanced endothelial cell migration. Unlike reference p17, the p17-Lyrm variant enhanced the activation of Akt and ERK, critical kinases in lymphomagenesis. p17-Lyrm clonogenic activity was dependent on the activation of Akt but not of ERK1/2. These results indicated that HIV p17 variants with distinct molecular signatures and functional properties may accumulate in lymphoid tissues of HIV-infected individuals where they may act as a local stimulus promoting the development of lymphomas.

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants.

BACKGROUND: HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships. METHODS: We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity. RESULTS: By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation. CONCLUSIONS: The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt. GENERAL SIGNIFICANCE: Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL.

Human lung epithelial cells support human metapneumovirus persistence by overcoming apoptosis.

Human metapneumovirus (hMPV) has been identified as a major cause of lower respiratory tract infection in children. Epidemiological and molecular evidence has highlighted an association between severe childhood respiratory viral infection and chronic lung diseases, such as asthma and chronic obstructive pulmonary disease. Currently, animal models have demonstrated the ability of hMPV to persist in vivo suggesting a role of the virus in asthma development in children. However, mechanisms involved in hMPV persistence in the respiratory tract are not yet understood. In the present study we monitored hMPV infection in human alveolar epithelial A549 cells in order to understand if the virus is able to persist in these cells upon acute infection. Our data show that hMPV initially induces an apoptotic process in A549 cells through poly (ADP-ribose) polymerase 1 cleavage, caspase-3/7 activation and Wee1 activity. The hMPV-infected cells were then able to overcome the apoptotic pathway and cell cycle arrest in G2/M by expressing B-cell lymphoma 2 and to acquire a reservoir cell phenotype with constant production of infectious virus. These findings provide evidence of the ability of hMPV to persist in alveolar epithelial cells and help in understanding the mechanisms responsible for hMPV persistence in the human respiratory tract.

HIV-1 matrix protein p17 and its variants promote human triple negative breast cancer cell aggressiveness.

BACKGROUND: The introduction of cART has changed the morbidity and mortality patterns affecting HIV-infected (HIV+) individuals. The risk of breast cancer in HIV+ patients has now approached the general population risk. However, breast cancer has a more aggressive clinical course and poorer outcome in HIV+ patients than in general population, without correlation with the CD4 or virus particles count. These findings suggest a likely influence of HIV-1 proteins on breast cancer aggressiveness and progression. The HIV-1 matrix protein (p17) is expressed in different tissues and organs of successfully cART-treated patients and promotes migration of different cells. Variants of p17 (vp17s), characterized by mutations and amino acid insertions, differently from the prototype p17 (refp17), also promote B-cell proliferation and transformation. METHODS: Wound-healing assay, matrigel-based invasion assay, and anchorage-independent proliferation assay were employed to compare the biological activity exerted by refp17 and three different vp17s on the triple-negative human breast cancer cell line MDA-MB 231. Intracellular signaling was investigated by western blot analysis. RESULTS: Motility and invasiveness increased in cells treated with both refp17 and vp17s compared to untreated cells. The effects of the viral proteins were mediated by binding to the chemokine receptor CXCR2 and activation of the ERK1/2 signaling pathway. However, vp17s promoted MDA-MB 231 cell growth and proliferation in contrast to refp17-treated or not treated cells. CONCLUSIONS: In the context of the emerging role of the microenvironment in promoting and supporting cancer cell growth and metastatic spreading, here we provide the first evidence that exogenous p17 may play a crucial role in sustaining breast cancer cell migration and invasiveness, whereas some p17 variants may also be involved in cancer cell growth and proliferation.

A single amino acid substitution confers B-cell clonogenic activity to the HIV-1 matrix protein p17.

Recent data highlight the presence, in HIV-1-seropositive patients with lymphoma, of p17 variants (vp17s) endowed with B-cell clonogenicity, suggesting a role of vp17s in lymphomagenesis. We investigated the mechanisms responsible for the functional disparity on B cells between a wild-type p17 (refp17) and a vp17 named S75X. Here, we show that a single Arginine (R) to Glycine (G) mutation at position 76 in the refp17 backbone (p17R76G), as in the S75X variant, is per se sufficient to confer a B-cell clonogenic potential to the viral protein and modulate, through activation of the PTEN/PI3K/Akt signaling pathway, different molecules involved in apoptosis inhibition (CASP-9, CASP-7, DFF-45, NPM, YWHAZ, Src, PAX2, MAPK8), cell cycle promotion and cancer progression (CDK1, CDK2, CDK8, CHEK1, CHEK2, GSK-3 beta, NPM, PAK1, PP2C-alpha). Moreover, the only R to G mutation at position 76 was found to strongly impact on protein folding and oligomerization by altering the hydrogen bond network. This generates a conformational shift in the p17 R76G mutant which enables a functional epitope(s), masked in refp17, to elicit B-cell growth-promoting signals after its interaction with a still unknown receptor(s). Our findings offer new opportunities to understand the molecular mechanisms accounting for the B-cell growth-promoting activity of vp17s.

Role of Autophagy in HIV-1 Matrix Protein p17-Driven Lymphangiogenesis.

AIDS-related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV-1-infected (HIV+) patients but does not affect the occurrence of ARLs to the same extent as that of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV-1 does not infect the neoplastic B cells, but HIV-1 proteins have been hypothesized to play a key role in sustaining a prolymphangiogenic microenvironment in lymphoid organs. The HIV-1 matrix protein p17 is detected in blood and accumulates in the germinal centers of lymph nodes of HIV+ patients under successful cART. The viral protein displays potent lymphangiogenic activity in vitro and in vivo This is, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin-1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here, we show that the p17 lymphangiogenic activity occurs on human lymph node-derived lymphatic endothelial cells (LN-LECs) under stress conditions only and relies entirely on activation of an autophagy-based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibits p17-triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy-incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor-driven lymphangiogenesis in HIV+ patients.IMPORTANCE AIDS-related lymphomas (ARLs) are the most common malignancies in HIV-1-infected (HIV+) patients after the introduction of combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV+ patients under successful cART. The HIV-1 matrix protein p17 is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several findings suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here, we show that p17 promotes lymphangiogenesis of human lymph node-derived lymphatic endothelial cells (LN-LECs). The lymphangiogenic activity of p17 is sustained by an autophagy-based pathway that enables LN-LECs to release prolymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.

Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17.

The human immune deficiency virus type 1 (HIV-1) matrix protein p17 (p17), although devoid of a signal sequence, is released by infected cells and detected in blood and in different organs and tissues even in HIV-1-infected patients undergoing successful combined antiretroviral therapy (cART). Extracellularly, p17 deregulates the function of different cells involved in AIDS pathogenesis. The mechanism of p17 secretion, particularly during HIV-1 latency, still remains to be elucidated. A recent study showed that HIV-1-infected cells can produce Gag without spreading infection in a model of viral latency. Here we show that in Gag-expressing cells, secretion of biologically active p17 takes place at the plasma membrane and occurs following its interaction with phosphatidylinositol-(4,5)-bisphosphate and its subsequent cleavage from the precursor Gag (Pr55Gag) operated by cellular aspartyl proteases. These enzymes operate a more complex Gag polypeptide proteolysis than the HIV-1 protease, thus hypothetically generating slightly truncated or elongated p17s in their C-terminus. A 17 C-terminal residues excised p17 was found to be structurally and functionally identical to the full-length p17 demonstrating that the final C-terminal region of p17 is irrelevant for the protein's biological activity. These findings offer new opportunities to identify treatment strategies for inhibiting p17 release in the extracellular microenvironment.

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens.

The rapid diagnosis of tuberculosis (TB) and the detection of drug-resistant Mycobacterium tuberculosis strains are critical for successful public health interventions. Therefore, TB diagnosis requires the availability of diagnostic tools that allow the rapid detection of M. tuberculosis and drug resistance in clinical samples. Here, we performed a multicenter study to evaluate the performance of the Seegene Anyplex MTB/NTM MDR-TB assay, a new molecular method based on a multiplex real-time PCR system, for detection of Mycobacterium tuberculosis complex (MTBC), nontuberculous mycobacteria (NTM), and genetic determinants of drug resistance. In total, the results for 755 samples (534 pulmonary and 221 extrapulmonary samples) were compared with the results of smears and cultures. For pulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 86.4% and 75.0%, respectively, and the specificities were 99% and 99.4%. For extrapulmonary specimens, the sensitivities of the Anyplex assay and acid-fast bacillus smear testing were 83.3% and 50.0%, respectively, and the specificities of both were 100%. The negative and positive predictive values of the Anyplex assay for pulmonary specimens were 97% and 100%, respectively, and those for extrapulmonary specimens were 84.6% and 100%. The sensitivities of the Anyplex assay for detecting isoniazid resistance in MTBC strains from pulmonary and extrapulmonary specimens were 83.3% and 50%, respectively, while the specificities were 100% for both specimen types. These results demonstrate that the Anyplex MTB/NTM MDR-TB assay is an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB and the detection of isoniazid resistance.

HIV-1 Matrix Protein p17 and its Receptors.

The HIV-1 matrix protein p17 (p17) plays a crucial role in the virus life cycle. It is released in the extracellular space from HIV-1-infected cells and accumulates in the tissues of patients, even in those successfully treated with highly active antiretroviral therapy. Extracellular p17 deregulates the biological functions of many different cells that are directly or indirectly implicated in AIDS pathogenesis. All p17 actions depend on interaction between its functional epitope (AT20), located at the protein N-terminal region, and different receptors expressed on target cells. This finding corroborates the importance of impeding p17/p17 receptors interaction as a contribution to block AIDS. In this article we review the interaction of p17 with heparan sulfate proteoglycans (HSPGs) and with the chemokine (C-X-C motif) receptor 1 (CXCR1) and 2 (CXCR2). We provide details on how p17 interacts with its receptors and how these interactions are central to the p17 biological activities. Moreover, we highlight the existence of a p17 variant, named S75X, which displays opposite effects on B-cell proliferation as compared to p17. A two-site model for p17 interaction with G-coupled receptors provides a possible explanation on how mutations naturally occurring within the primary amino acid structure can lead S75X to activate the Akt signaling pathway and to promote B-cell growth and transformation. Identification of p17 interaction with HSPGs, CXCR1 and CXCR2 as a fundamental event in supporting its activity could help to find new treatment approaches aimed at blocking all p17/p17 receptors interactions and, consequently, p17 detrimental activities.

Role of HIV-1 matrix protein p17 variants in lymphoma pathogenesis.

Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.

Long-lasting humoral immune response induced in HIV-1-infected patients by a synthetic peptide (AT20) derived from the HIV-1 matrix protein p17 functional epitope.

OBJECTIVE: A therapeutic vaccination based on a synthetic peptide (AT20) representative of the HIV-1 matrix protein p17 (p17) functional region, coupled to keyhole limpet hemocyanin (KLH) AT20-KLH was capable of inducing the production of high-avidity antibodies (Abs) toward a previous untargeted p17 hotspot of functional activity in highly active antiretroviral therapy (HAART)-treated HIV-1-infected patients. Since avidity of Abs after immunization and the retention of antigens are important in sustaining the long-lasting production of specific humoral responses, we asked whether AT20-KLH vaccination would result in development of a long-lived immune response. METHODS: The long-term duration of Ab response to AT20-KLH has been evaluated in 10 patients previously enrolled for the AT20-KLH vaccination trial at day 898 post-immunization. Ab titer and their avidity was assessed using specifically designed ELISA assays, whereas their neutralizing capacity was estimated in vitro using a 'wound sealing assay'. RESULTS: Data obtained show that high titers of specific anti-AT20 Abs were maintained at more than 2 years after the last immunization. Furthermore, these Abs were capable to neutralize exogenous p17, as assessed by ability of sera derived from AT20-KLH-immunized patients to block the ability of p17 to promote cell migration in vitro. CONCLUSION: This finding attests for a successful AT20-KLH vaccine molecule formulation and for an effective HAART-dependent Ab persistence.