Signal strength controls the rate of polarization within CTLs during killing.

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca2+ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.

Centrosome dysfunction: a link between senescence and tumor immunity.

Centrosome aberrations are hallmarks of human cancers and contribute to the senescence process. Structural and numerical centrosome abnormalities trigger mitotic errors, cellular senescence, cell death, genomic instability and/or aneuploidy, resulting in human disorders such as aging and cancer and affecting immunity. Interestingly, centrosome dysfunction promotes the secretion of multiple inflammatory factors that act as pivotal drivers of senescence and tumor immune escape. In this review, we summarize the forms of centrosome dysfunction and further discuss recent advances indicating that centrosome defects contribute to acceleration of senescence progression and promotion of tumor cell immune evasion in different ways.

NANOG/NANOGP8 Localizes at the Centrosome and is Spatiotemporally Associated with Centriole Maturation.

NANOG is a transcription factor involved in the regulation of pluripotency and stemness. The functional paralog of NANOG, NANOGP8, differs from NANOG in only three amino acids and exhibits similar reprogramming activity. Given the transcriptional regulatory role played by NANOG, the nuclear localization of NANOG/NANOGP8 has primarily been considered to date. In this study, we investigated the intriguing extranuclear localization of NANOG and demonstrated that a substantial pool of NANOG/NANOGP8 is localized at the centrosome. Using double immunofluorescence, the colocalization of NANOG protein with pericentrin was identified by two independent anti-NANOG antibodies among 11 tumor and non-tumor cell lines. The validity of these observations was confirmed by transient expression of GFP-tagged NANOG, which also colocalized with pericentrin. Mass spectrometry of the anti-NANOG immunoprecipitated samples verified the antibody specificity and revealed the expression of both NANOG and NANOGP8, which was further confirmed by real-time PCR. Using cell fractionation, we show that a considerable amount of NANOG protein is present in the cytoplasm of RD and NTERA-2 cells. Importantly, cytoplasmic NANOG was unevenly distributed at the centrosome pair during the cell cycle and colocalized with the distal region of the mother centriole, and its presence was markedly associated with centriole maturation. Along with the finding that the centrosomal localization of NANOG/NANOGP8 was detected in various tumor and non-tumor cell types, these results provide the first evidence suggesting a common centrosome-specific role of NANOG.

PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome.

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.

Actin clearance promotes polarized dynein accumulation at the immunological synapse.

Immunological synapse (IS) formation between a T cell and an antigen-presenting cell is accompanied by the reorientation of the T cell centrosome toward the interface. This polarization response is thought to enhance the specificity of T cell effector function by enabling the directional secretion of cytokines and cytotoxic factors toward the antigen-presenting cell. Centrosome reorientation is controlled by polarized signaling through diacylglycerol (DAG) and protein kinase C (PKC). This drives the recruitment of the motor protein dynein to the IS, where it pulls on microtubules to reorient the centrosome. Here, we used T cell receptor photoactivation and imaging methodology to investigate the mechanisms controlling dynein accumulation at the synapse. Our results revealed a remarkable spatiotemporal correlation between dynein recruitment to the synaptic membrane and the depletion of cortical filamentous actin (F-actin) from the same region, suggesting that the two events were causally related. Consistent with this hypothesis, we found that pharmacological disruption of F-actin dynamics in T cells impaired both dynein accumulation and centrosome reorientation. DAG and PKC signaling were necessary for synaptic F-actin clearance and dynein accumulation, while calcium signaling and microtubules were dispensable for both responses. Taken together, these data provide mechanistic insight into the polarization of cytoskeletal regulators and highlight the close coordination between microtubule and F-actin architecture at the IS.

Anti-centrosome antibodies: Prevalence and disease association in Chinese population.

Anti-centrosome antibodies are rare findings with undefined clinical significance in clinical research. We aimed at investigating the prevalence and clinical significance of anti-centrosome antibodies in Chinese population. Testing results of total of 281,230 ANA-positive sera were retrospectively obtained from West China Hospital Sichuan University in China between 2008 and 2017. We retrospectively collected and analysed the clinical and laboratory data of the patients with positive anti-centrosome antibody. Of the 356 453 patients tested, 281 230 patients had positive antinuclear antibodies (ANAs, 78.9%), but only 78 patients with positive anti-centrosome antibodies (0.022%), of which 74.4% are females. Diagnoses were established in 69 of 78 patients: 37 cases were autoimmune diseases, mainly including undifferentiated connective tissue diseases (UCTD, 9/37), rheumatoid arthritis (RA, 6/37), Sjögren's syndrome (SS, 5/37) and primary biliary cirrhosis (PBC, 5/37), and the remaining were other autoimmune conditions. The most frequent clinical symptoms of the anti-centrosome-positive patients were arthralgia and eyes and mouth drying. Additionally, 86.7% of anti-centrosome antibodies were not associated with other ANA profiles; however, when associated, the most frequent ANA was anti-U1RNP. Anti-centrosome antibodies are featured by a low prevalence and female gender predominance. They are correlated with some specific diseases, both autoimmune diseases, especially UCTD, RA, SS and PBC, and non-autoimmune diseases, such as infection and cancer, which suggests that they might be potential supporting serological markers of these diseases.

Simulations of centriole of polarized centrosome as a monopole antenna in immune and viral synapses.

The immune synapse (IS) is a temporary interface between an antigen-presenting cell and an effector lymphocyte. Viral synapse is a molecularly organized cellular junction that is structurally similar to the IS. Primary cilium is considered as a functional homologue of the IS due to the morphological and functional similarities in architecture between both micotubule structures. It has been hypothesized that endogenous electromagnetic field in the cell is generated by a unique cooperating system between mitochondria and microtubules. We are extending this prior hypothesis of the endogenous electromagnetic field in the cell postulating that polarized centriole in immune and viral synapse could serve as a monopole antenna. This is an addition to our hypothesis that primary cilium could serve as a monopole antenna. We simulated the distribution of electric field of centriole of polarized centrosome as a monopole antenna in immune and viral synapse. Very weak electromagnetic field of polarized centriole of CD8+ T lymphocyte in IS can contribute to the transport of cytolytic granules into the attacked (cancer) cell. Analogically, very weak electromagnetic field of polarized centriole in viral synapse of infected CD4 cells can aid the transport of viruses (human immunodeficiency virus) to non-infected CD4 cells. We hypothesized that healthy organisms need these monopole antennas. If, during the neoplastic transformation, healthy cells lose monopole antennas in form of primary cilia, the IS aims to replace them by monopole antennas of polarized centrioles in IS to restore homeostasis.

Expression of genes encoding centrosomal proteins and the humoral response against these proteins in chronic myeloid leukemia.

As the extent of centrosome abnormalities in chronic myeloid leukemia (CML) correlates with disease stage and karyotype alterations, abnormal expression of genes encoding centrosomal proteins may be an early prognostic marker of disease progression. In the present study, we showed that in comparison with healthy controls, the expression of four centrosomal genes (AURKA, HMMR, PLK1 and ESPL1) in the peripheral blood of CML patients was significantly enhanced at diagnosis and decreased to the basal level in most patients treated with imatinib mesylate for three months. In the remaining patients (17%), this decrease was delayed and was associated with worse overall survival. The detection of antibodies in sera showed that patients with higher overall antibody production had superior outcomes in terms of achieving major molecular response and failure-free survival. These data suggest that the dynamics of the response of centrosomal genes should be considered as a risk factor and immunity against centrosomal proteins may contribute to treatment response.

Autoantibodies in breast cancer sera are not epiphenomena and may participate in carcinogenesis.

BACKGROUND: The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases. METHODS: We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens. RESULTS: Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative. CONCLUSIONS: The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

Anti-centrosome antibodies in breast cancer are the expression of autoimmunity.

Centrosome abnormalities have been observed in nearly all human solid tumors, but their role in tumorigenesis is unclear. We have demonstrated that autoantibodies reacting with antigens in centrosomes are frequently found in BC sera. In this work, we attempted to characterize the centrosome antigens associated with BC. We immunoscreened a T7 cDNA library of BC proteins with BC sera, and the autoantigens identified were printed as a microarray and hybridized with BC and control sera. We used immunohistochemistry (IHC) to investigate the expression of the cloned autoantigens in BC tissue. Immunoscreening with BC sera led to the cloning of autoantibodies recognizing epitopes developing in a family of proteins located on centrosomes such as peri-centriolar material-1, isomorph CRA, stathmin1, HS actin gamma1, SUMO/sentrin peptidase 2, and ubiquitin-conjugating enzyme E2 variant 1. Antibody reactivity to these proteins that are associated with centrosome assembly and/or microtubule function was highly associated with the diagnosis of BC. IHC staining of formalin-fixed paraffin-embedded sections with specific antibodies showed that aurora and stathmin are expressed in BC. The discovery of autoantibodies to important centrosome antigens associated with BC suggests that this immune reactivity could be related to autoimmunity developing in BC. Our finding that some of these antibodies are also present in a group of healthy women suggests that breakdown of tolerance to centrosome proteins may occur early in breast carcinogenesis and that autoantibodies to centrosome antigens might be biomarkers of early BC.

Centrosomal localisation of the cancer/testis (CT) antigens NY-ESO-1 and MAGE-C1 is regulated by proteasome activity in tumour cells.

The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-1(91-150) and MAGE-C1(900-1116) were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.

The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.

Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

Deterministic mechanical model of T-killer cell polarization reproduces the wandering of aim between simultaneously engaged targets.

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell-cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell-cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation.

Novel centrosome protein, TCC52, is a cancer-testis antigen.

A novel centrosome protein, TCC52, was identified as a cancer-testis (CT) antigen. The TCC52 gene was tissue-restricted in normal tissues but highly expressed in lung cancer tissues and some cancer cell lines. Immunoglobulin G antibody specific to TCC52 was detected in serum samples from patients with prostate cancer (59.4%, 69/116), cholangiocarcinoma (17.6%, 6/34), laryngeal cancer (8%, 8/100) and lung cancer (5.6%, 4/71) in patients, rather than from healthy donors. Based on its restricted expression pattern and immunogenicity in some types of tumor, TCC52, as a novel CT antigen, would be a promising candidate for cancer immunotherapy.

PP2A-dependent disruption of centrosome replication and cytoskeleton organization in Drosophila by SV40 small tumor antigen.

Viruses of the DNA tumor virus family share the ability to transform vertebrate cells through the action of virus-encoded tumor antigens that interfere with normal cell physiology. They accomplish this very efficiently by inhibiting endogenous tumor suppressor proteins that control cell proliferation and apoptosis. Simian virus 40 (SV40) encodes two oncoproteins, large tumor antigen, which directly inhibits the tumor suppressors p53 and Rb, and small tumor antigen (ST), which interferes with serine/threonine protein phosphatase 2A (PP2A). We have constructed a Drosophila model for SV40 ST expression and show that ST induces supernumerary centrosomes, an activity we also demonstrate in human cells. In early Drosophila embryos, ST also caused increased microtubule stability, chromosome segregation errors, defective assembly of actin into cleavage furrows, cleavage failure, a rise in cyclin E levels and embryonic lethality. Using ST mutants and genetic interaction experiments between ST and PP2A subunit mutations, we show that all of these phenotypes are dependent on ST's interaction with PP2A. These analyses demonstrate the validity and utility of Drosophila as a model for viral oncoprotein function in vivo.

Autoantibodies to mitotic apparatus: association with other autoantibodies and their clinical significance.

The most important mitotic apparatus (MA) antigens are centrosome (CE), nuclear mitotic apparatus (NuMA-1, NuMA-2), midbody, and centromere F (CENP-F). We studied associations of anti-MA antibodies with other autoantibodies and their clinical significance. A total of 6270 patients were studied for the presence of anti-MA antibodies on HEp-2 cells. Sera positive for anti-MA were tested for anti-extractable nuclear antigens (ENA) antibodies. Anti-MA antibodies were detected in 56 (45 females and 11 males) of 6270 sera (0.9%). Of these 56, NuMA-1 was found in 23, NuMA-2 in 7, CE in 20, CENP-F in 5, and CENP-F/centrosome in 1 case. Anti-NuMA-1 were associated with anti-ENA antibodies (p < 0.001). Diagnoses were established in 43/56 patients: 22 connective tissue diseases, 7 infections, 6 autoimmune hepatitis, 3 vasculitis, 3 primary antiphospholipid syndrome, 1 malignancy, and 1 fever of unknown origin. The differential diagnosis of anti-NuMA-1-positive patients must include Sjögren's syndrome, while patients with anti-CE antibodies must be observed for HCV infection.

Spastin interacts with the centrosomal protein NA14, and is enriched in the spindle pole, the midbody and the distal axon.

Hereditary spastic paraplegia (HSP) is characterized by the specific retrograde degeneration of the longest axons in the central nervous system, the corticospinal tracts. The gene most frequently involved in autosomal dominant cases of this disease, SPG4, encodes spastin, an ATPase belonging to the AAA family. AAA proteins are thought to exert their function by the energy-dependent rearrangement of protein complexes. The composite function of these proteins is directed by their binding to regulatory factors and adaptor proteins that target their activity into specific pathways in vivo. We previously found that overexpressed spastin interacts dynamically with microtubules and displays microtubule-severing activity. Here, we demonstrate that spastin is enriched in cell regions containing dynamic microtubules. During cell division spastin is found in the spindle pole, the central spindle and the midbody, whereas in immortalized motoneurons it is enriched in the distal axon and the branching points. Furthermore, spastin interacts with the centrosomal protein NA14, and co-fractionates with gamma-tubulin, a centrosomal marker. Deletion of the region required for binding to NA14 disrupts spastin interaction with microtubules, suggesting that NA14 may be an important adaptor to target spastin activity at the centrosome. These data strongly argue that spastin plays a role in cytoskeletal rearrangements and dynamics, and provide an attractive explanation for the degeneration of motor axons in HSP.

Spectrum of centrosome autoantibodies in childhood varicella and post-varicella acute cerebellar ataxia.

BACKGROUND: Sera from children with post-varicella infections have autoantibodies that react with centrosomes in brain and tissue culture cells. We investigated the sera of children with infections and post-varicella ataxia and related conditions for reactivity to five recombinant centrosome proteins: gammagamma-enolase, pericentrin, ninein, PCM-1, and Mob1. METHODS: Sera from 12 patients with acute post-varicella ataxia, 1 with post-Epstein Barr virus (EBV) ataxia, 5 with uncomplicated varicella infections, and other conditions were tested for reactivity to cryopreserved cerebellum tissue and recombinant centrosome proteins. The distribution of pericentrin in the cerebellum was studied by indirect immunofluorescence (IIF) using rabbit antibodies to the recombinant protein. Antibodies to phospholipids (APL) were detected by ELISA. RESULTS: Eleven of 12 children with post-varicella ataxia, 4/5 children with uncomplicated varicella infections, 1/1 with post-EBV ataxia, 2/2 with ADEM, 1/2 with neuroblastoma and ataxia, and 2/2 with cerebellitis had antibodies directed against 1 or more recombinant centrosome antigens. Antibodies to pericentrin were seen in 5/12 children with post-varicella ataxia but not in any of the other sera tested. IIF demonstrated that pericentrin is located in axons and centrosomes of cerebellar cells. APL were detected in 75% of the sera from children with post-varicella ataxia and 50% of children with varicella without ataxia and in none of the controls. CONCLUSION: This is the first study to show the antigen specificity of anti-centrosome antibodies in children with varicella. Our data suggest that children with post-varicella ataxia have unique autoantibody reactivity to pericentrin.

Enhancing major histocompatibility complex class I antigen presentation by targeting antigen to centrosomes.

Several strategies that increase proteasomal degradation of antigen have been shown to improve MHC class I presentation of antigen. Because recent studies have demonstrated that the centrosome is a subcellular compartment rich in proteasomes, we hypothesized that targeting a tumor antigen to centrosomal compartments would enhance both the MHC class I presentation of antigen and the vaccine potency. We, therefore, created a chimera of gamma-tubulin, an established centrosomal marker, with a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, in a DNA vaccine. The linkage of gamma-tubulin to E7-targeted antigen to centrosomal compartments, resulted in enhanced MHC class I presentation of E7, and led to a marked increase in the number of E7-specific CD8(+) T-cell precursors as well as a potent CD4-independent antitumor effect against an E7-expressing tumor cell line, TC-1. In addition, vaccination with gamma-tubulin/E7 DNA in transporter associated with antigen presentation (TAP)-1-knockout mice revealed that the enhancement of E7-specific CD8(+) T-cell immune responses is TAP-1-dependent. Our data suggest that the centrosome may be an important locus for MHC class I antigen processing and that targeting antigen to the centrosome can improve DNA vaccine potency.

[Clinical significance of fluoroscopic patterns specific for the mitotic spindle in patients with rheumatic diseases]. / Significato clinico dei quadri fluoroscopici specifici per il fuso mitotico in pazienti affetti da malattie reumatiche.

OBJECTIVE: To determine the clinical significance of anti-NuMA and anti-HsEg5 antibodies in a group of patients affected with rheumatic diseases. MATERIALS AND METHODS: Indirect immunofluorescence on HEp-2000 cells at serum dilution of 1:40 was used to examine 26 sera which had previously showed a "mitotic spindle" fluoroscopic pattern type during laboratory routine. RESULTS: 21 sera (80,7%) were identified with NuMA and 5 (19,3%) with HsEg5 patterns alone or associated with other ANA patterns. However only patients with isolated positivity and that is 15 with NuMA and 4 with HsEg5 stainings were included in this study. Of the NuMA positive patients 5 were affected with arthropathies associated to different forms of thyroiditis, 2 with seronegative arthritis, 2 with antiphospholipid syndrome, 1 with systemic lupus erythematosus (SLE), 1 with rheumatoid arthritis, 1 with sicca syndrome, 1 with undifferentiated connective tissue disease, 1 with Mycoplasma pneumoniae infection and 1 with retinal thrombosis. Of the HsEg5 positive patients 3 were affected with SLE and 1 with seronegative arthritis. CONCLUSIONS: NuMA does not prevail in any defined rheumatic disease, while HsEg5 staining were more frequent (75%) in patients affected with SLE all of whom showing high antibody titres.