Activation of plasmacytoid dendritic cells by apoptotic particles - mechanism for the loss of immunological tolerance in Sjögren's syndrome.

Sjögren's syndrome (SS) is a common autoimmune disease targeting salivary and lacrimal glands. It is strongly female-dominant, characterized by low oestrogen levels combined with a local intracrine dihydrotestosterone defect. We hypothesized that these hormonal deficits lead to increased apoptosis of the epithelial cells and plasmacytoid dendritic cell (pDC)-mediated proinflammatory host responses. Expression of Toll-like receptors (TLRs)-7 and -9 and cytokine profiles was studied in pDCs treated with apoptotic particles collected in consecutive centrifugation steps of media from apoptotic cells. Expression and localization of SS autoantigens in these particles was also analysed. Furthermore, the effects of sex steroids were studied in pDCs cultured with several concentrations of dihydrotestosterone and 17-ß-oestradiol, and in saliva of patient treated with dehydroepiandrosterone. Apoptosis of the epithelial cells led to cleavage and translocation of SS-autoantigens, α-fodrin and SS-A, into apoptotic particles. The apoptosis-induced apoptotic particles also contained another SS-autoantigen, hy1-RNA. These particles were internalized by pDCs in a size-dependent manner and affected TLR-7 and -9 expression and the production of proinflammatory cytokines. The analysed androgens protected cells from apoptosis, influenced redistribution of autoantigens and diminished the apoptotic particle-stimulated increase of the TLRs in pDCs. Our findings suggest that the formation of apoptotic particles may play a role in loss of immune tolerance, manifested by production of autoantibodies and the onset of autoinflammation in SS.

Histamine metabolism and transport are deranged in human keratinocytes in oral lichen planus.

BACKGROUND: Recent reports have indicated that nonimmune cells can produce low concentrations of histamine. This observation, together with the discovery of the high-affinity histamine H4 receptor (H4 R), has added additional layers of complexity to our understanding of histamine signalling. Human oral keratinocytes (HOKs) possess a uniform H4 R pattern, which is deranged in oral lichen planus (OLP). OBJECTIVES: To investigate histamine metabolism and transport in HOKs of healthy controls and patients with OLP. METHODS: Tissue sections and cultured primary HOKs were studied using immunostaining, quantitative real-time polymerase chain reaction and confocal microscopy. Histamine levels were analysed using high-performance liquid chromatography. RESULTS: l-histidine decarboxylase (HDC) and organic cation transporter (OCT)3 were increased in mRNA and protein levels in patients with OLP compared with controls. In contrast, histamine N-methyltransferase (HNMT) immunoreactivity was decreased in OLP. OCT1/OCT2 and diamine oxidase were not detectable in either tissue sections or in HOKs. Immunolocalization of HDC and OCT3 in HOKs revealed moderate-to-high expression within cytoplasm and cell boundaries. Stimulation with lipopolysaccharide (LPS) or interferon-γ upregulated HDC-gene transcript in HOKs, whereas this was downregulated with high histamine concentration and tumour necrosis factor-α. LPS induced a dose-dependent release of low histamine in HOKs, while high histamine concentration downregulated epithelial adhesion proteins. CONCLUSIONS: HOKs are histamine-producing cells. They release histamine via OCT3 channels in concentrations too low to activate the classical low-affinity H1 R and H2 R, but high enough to stimulate the high-affinity H4 R in autocrine and paracrine modes. The substantially deranged histamine metabolism and transport in OLP could, in part, contribute to the disease pathogenesis.

Diagnosis, gB genotype distribution and viral load of symptomatic congenitally infected CMV patients in Cuba.

OBJECTIVE: Cytomegalovirus (CMV) is the leading cause of viral congenital infection. Some viral factors have been proposed to be CMV pathogenicity markers. The objective of this study was to investigate the frequency of congenital CMV infection in symptomatic patients and the possible association with the CMV glycoprotein B (gB) genotype and viral load. STUDY DESIGN: A total of 361 newborns (NB) and 158 pregnant women (PW) with clinically suspected CMV infection were enrolled. Studied samples included urine, saliva, serum, vaginal swabs and amniotic fluid. CMV infection was diagnosed by multiplex nested PCR. CMV gB genotyping was performed on infected samples, followed by viral load determination. RESULTS: Overall, 18.7% of the tested patients were positive for CMV infection, 19.7% of NB were congenitally infected and 16.5% of PW showed active CMV infection. gB-2 was the most prevalent genotype detected (39/97 patients). gB CMV mixed infections were detected in 12 patients. gB-2 was associated with mono-infections (P<0.01). Mixed infections showed higher levels of viral load compared with gB mono-infection (P=0.03). Hepatomegaly, splenomegaly, jaundice, sepsis-like syndrome and malformations were the most prevalent clinical findings. gB-4 was more frequently associated with sepsis-like syndrome than other gB genotypes (P=0.04, odds ratio=4.3, confidence interval: 0.9 to 21.6). The difference in medians of CMV load was statistically significant among patients presenting different clinical signs (P=0.04). CONCLUSIONS: This study showed that CMV is a frequent cause of congenital infection in symptomatic Cuban patients. Despite gB2 being the most frequently detected, gB-4 was the only genotype associated with clinical features (sepsis-like syndrome in NB). No other associations among specific genotypes and clinical characteristics were found. Further studies are needed to clarify the role that viral load and genotype play in the outcome of congenital infection.

Diminished salivary epidermal growth factor secretion: a link between Sjögren's syndrome and autoimmune gastritis?

OBJECTIVES: Healthy human labial salivary glands produce epidermal growth factor (EGF). In Sjögren's syndrome (SS), EGF staining is diminished. SS is also associated with chronic autoimmune corpus gastritis. We therefore hypothesized that EGF secretion would be diminished in SS and that this could affect gastric target cells. METHODS: Salivary EGF secretion in SS was compared to that in healthy controls using an enzyme-linked immunosorbent assay (ELISA). EGF receptor (EGFR) immunoreactive cells in the gastric corpus of healthy human subjects were analysed using immunostaining. RESULTS: Salivary secretion of EGF was diminished in SS patients (232.4, range 52.6-618.4, vs. 756.6, range 105.3-1631.6 pg/min, p = 0.002). Proton-pump positive parietal cells were mostly EGFR immunoreactive whereas very few pepsinogen I (PGI)-positive cells were EGFR positive. CONCLUSIONS: As EGF is relatively acid resistant, salivary gland-derived EGF might participate in an exo/endocrine mode of parietal cell maintenance in the gastric corpus. Deficiency of salivary gland-derived EGF in SS patients may cause impairment of gastric parietal cells resulting in exposure of immunogenic cryptic antigens and loss of immunological self-tolerance.

Isolation of Mycoplasma genitalium from patients with urogenital infections: first report from the Latin-American region.

Isolation of Mycoplasma genitalium from clinical specimens remains difficult. We describe a modified culture system based on Vero cells grown in medium 199 with 2% foetal bovine serum (FBS). The culture system was evaluated using early passage M. genitalium strains M6271 and M6311 with growth monitoring by quantitative TaqMan PCR. Eleven endocervical swabs and one male urethral swab positive by 16S rRNA and MgPa1-3 PCRs were quantified and inoculated into Vero cell suspensions in medium 199 supplemented with 2% FBS and antibiotics. Cultures were incubated for 14 days. Cell passages and growth monitoring by TaqMan PCR were performed until the growth of M. genitalium reached ≥10(6) geq/mL. Confirmation of the new M. genitalium strains was performed by sequencing a 281 bp fragment of mgpB. The growth of Mycoplasma genitalium strains was recorded for all urogenital swab specimens in the modified cell-culture system. Growth of M. genitalium was obtained within 2 months and yielded 12 M. genitalium strains with all 11 isolates from females of an identical, but unique genotype. To our knowledge, this is the first successful isolation of M. genitalium in the Latin-American region. The use of Vero cell culture in 199 medium with 2% FBS is a method comparable to the Ultroser G culture system for isolation of M. genitalium. Genotyping of clinical samples and isolates should be performed to document the absence of cross-contamination.

HCMV seroprevalence and associated risk factors in pregnant women, Havana City, 2007 to 2008.

OBJECTIVE: To prenatally identify pregnant women at risk of developing congenital infection due to human cytomegalovirus (HCMV). METHODS: One thousand one hundred and thirty-one pregnant women from three municipalities from Havana City were serologically screened for HCMV infection (IgM/IgG, IgG avidity) from January 2007 to January 2008. Demographical, epidemiological, and clinical variables were correlated to serologic status to identify predictors of seroconversion in pregnancy. RESULTS: The majority of women were seropositive to HCMV (92.6%); 27 women (2.4%) developed HCMV active infection during pregnancy, defined by the detection of IgG+ and IgM+ (7 women), IgM+ and IgG- (2 women), and IgG seroconversion (18 women). Susceptibility of active HCMV infection during pregnancy was associated with maternal age < 20 years and nulligravidity. Primary infection was detected in 20 pregnant women (1.8%), whereas 7 patients (0.6%) had active non-primary infection. CONCLUSION: Although pregnant women in Cuba have high seroprevalence rates for HCMV, those younger than 20 years and nulligravidae are at risk of acquiring infection during pregnancy.

High mobility group box-1 (HMGB-1) in osteoarthritic cartilage.

OBJECTIVES: Nucleosomal high mobility group box-1 (HMGB-1) is translocated and released from necrotic and activated cells as an endogenous danger signal (alarmin) and cytokine. It was hypothesised that it plays a role in osteoarthritis (OA). characterised by cellular activation, inflammation and enchondral bone formation. METHODS: Bovine knee joint samples, collected from culled animals, were scored using histologic/histochemical grading to intact looking, mild, moderate or severe and immunohistochemically stained for HMGB-1. Chondrocyte pellets, produced from human bone marrow-derived mesenchymal stem cells and stimulated with tumour necrosis factor-a (TNF-alpha), were similarly stained. RESULTS: In healthy looking OA cartilage chondrocyte nuclei were usually HMGB-1 negative and in mild OA staining was restricted to nuclei. In moderate OA lesions HMGB-1 was also seen in the cytoplasm and occasionally pericellular matrix and in severe OA lesions often also in intra- and inter-territorial matrix. The tidemark in healthy cartilage did not contain HMGB-1, which however was seen at this interface as linear deposits even in intact-looking and mild OA lesions, as multiple wave-like deposits in moderate and as heavy granular deposits in severe lesions. TNF-alpha stimulation of chondrocytes caused translocation of HMGB-1 from the nucleus to the cytoplasm. CONCLUSIONS: In resting chondrocytes tight nucleosomal HMGB-1 binding might cause steric hindrance of immunostaining. TNF-alpha- or OA-mediated activation leads to nuclear staining and cytoplasmic translocation. Advancing OA leads to increasingly intense extra-/pericellular deposition of HMGB-1 alarmin, indicating local chondrocyte activation and/or necrosis. In particular, HMGB-1 at the tidemark might play a role in the pathological thickening of subchondral bone plate/osteophyte formation.

Pro-inflammatory, pleiotropic, and anti-inflammatory TNF-alpha, IL-6, and IL-10 in experimental porcine intervertebral disk degeneration.

The aim of this study was to check the balance between tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-10 (IL-10) in well-developed end-stage disk disease in the disk itself as well as in paradiskal spine. In 6 domestic pigs the cranial bony end plate of the L4 vertebra was perforated to the nucleus pulposus. At 3 months the degenerated experimental and contiguous control disks, together with the adjoining bony and cartilaginous vertebral end plates, bone marrow, and spinal ligaments, were excised and used for immunohistochemical analysis. In general, there were more TNF-alpha and in particular IL-10 positive cells in the degenerated disks than in the control disks, whereas the number of IL-6 labeled cells did not differ among sites or between control and experimental intervertebral disks. These results suggest that TNF-alpha and IL-10 are involved in the late reparatory phases of the experimental disk lesion. Use of an experimental model showed that strictly disk-directed manipulation and degeneration are also reflected in the contiguous vertebrae, including adjoining cartilage, bone, marrow, and ligaments.

Type IV collagen alpha-chain composition in synovial lining from trauma patients and patients with rheumatoid arthritis.

OBJECTIVE: Normal synovial lining is composed of macrophage-like type A and fibroblast-like type B lining cells. This sheet-like structure lacks a basement membrane, but its intercellular substance contains some basement membrane components, including type IV collagen. We undertook this study to determine the alpha-chain composition of type IV collagen in normal and arthritic synovial lining, using monoclonal alpha-chain antibodies. METHODS: Samples were analyzed using avidin-biotin-peroxidase complex staining for the presence of collagen alpha1/2(IV), alpha3(IV), alpha4(IV), alpha5(IV), alpha6(IV), matrix metalloproteinase 2 (MMP-2), and MMP-9, and the enzyme activity was detected using gelatin zymography. Double immunofluorescence was performed for type IV collagen/MMP-9 and type IV collagen/CD68. Synovial fibroblasts were studied using quantitative reverse transcriptase-polymerase chain reaction. RESULTS: In mildly inflamed synovium from 5 trauma patients, alpha1/2(IV) chains were strongly stained, but alpha5(IV) and alpha6(IV) chains were weakly stained. Coding messenger RNA was shown in cultured synovial fibroblasts. Basement membranes of blood vessels contained all alpha(IV) chains and served as useful positive sample controls. In the synovial lining from 5 patients with rheumatoid arthritis (RA), all alpha-chains were absent/very weakly stained. This was coupled with numerous type A lining cells containing MMP-9 (type IV collagenase), also found in synovial fluid. CONCLUSION: Synovial lining has a unique and very limited alpha-chain composition, different from that of the vascular basement membrane, which contains all alpha-chains. This special composition and lack of nidogen are probably of relevance for the bidirectional translining diffusion. Such tentative alpha-chain-dependent adhesive and transport-regulating properties seem to be deranged in RA, probably in part due to type IV collagenases produced in the lining and/or released by transmigrating or synovial fluid neutrophils.

Human immunodeficiency virus and other sexually transmitted diseases in Cuban women.

A cross-sectional study was performed in 60 Cuban women of child-bearing age who were seropositive for human immunodeficiency virus (HIV) and 60 controls. Human papillomavirus (HPV) was identified most frequently, with oncogenic HPV serotypes 16, 33 and 58 detected in HIV-positive patients, and serotypes 11, 33 and 51 in the controls (relative risk 4.41; 95% CI 2.21-8.29). Syphilis and hepatitis B and C viruses were detected exclusively in HIV-seropositive women (p<0.05). Sexually transmitted diseases (STDs) appeared to pose a substantial health problem, especially for HIV-positive women. Clinics should consider screening and treatment for STDs as part of their HIV prevention programmes.

[Meningoencephalitis caused by Herpesviridae family virus in Cuba. 1996-1998]. / Meningoencefalitis por virus de la familia Herpesviridae en Cuba. Período 1996-1998.

Herpesvirus isolation from cerebrospinal fluid (CSF) has proven not to be very useful because of its low sensitivity; that is the reason why PCR has considerably improved the diagnosis of several viral infections of the central nervous system. We studied, by means of PCR, 253 CSF received in our laboratory from 1996 to 1998, which had a suspected diagnosis of herpesviral-associated neurologic disease (encephalitits or meningoencephalitis). Of these, 182 were from HIV seropositive patients and the other 71 were from HIV seronegative. The percentage of all positive cases was 22.5% in AIDS patients and CMV was the virus most frequently detected (13.7%). We detected a percentage of 29.5 of positive cases in immunocompetent individuals, being HSV the virus most frequently found. Other herpesviruses (varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6) were also detected in some cases in both groups. Finally, we obtained clinical data from all positive patients and established a relationship with PCR results.

Meningoencefalitis por virus de la familia Herpesviridae en Cuba. Período 1996-1998 / Meningoencephalitis caused by Herpesviridae family virus in Cuba. 1996-1998

Herpesvirus isolation from cerebrospinal fluid (CSF) has proven not to be very useful because of its low sensitivity; that is the reason why PCR has considerably improved the diagnosis of several viral infections of the central nervous system. We studied, by means of PCR, 253 CSF received in our laboratory from 1996 to 1998, which had a suspected diagnosis of herpesviral-associated neurologic disease (encephalitits or meningoencephalitis). Of these, 182 were from HIV seropositive patients and the other 71 were from HIV seronegative. The percentage of all positive cases was 22.5 in AIDS patients and CMV was the virus most frequently detected (13.7). We detected a percentage of 29.5 of positive cases in immunocompetent individuals, being HSV the virus most frequently found. Other herpesviruses (varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6) were also detected in some cases in both groups. Finally, we obtained clinical data from all positive patients and established a relationship with PCR results.(AU)

Meningoencefalitis por virus de la familia Herpesviridae en Cuba. Período 1996-1998 / Meningoencephalitis caused by Herpesviridae family virus in Cuba. 1996-1998

Herpesvirus isolation from cerebrospinal fluid (CSF) has proven not to be very useful because of its low sensitivity; that is the reason why PCR has considerably improved the diagnosis of several viral infections of the central nervous system. We studied, by means of PCR, 253 CSF received in our laboratory from 1996 to 1998, which had a suspected diagnosis of herpesviral-associated neurologic disease (encephalitits or meningoencephalitis). Of these, 182 were from HIV seropositive patients and the other 71 were from HIV seronegative. The percentage of all positive cases was 22.5 in AIDS patients and CMV was the virus most frequently detected (13.7). We detected a percentage of 29.5 of positive cases in immunocompetent individuals, being HSV the virus most frequently found. Other herpesviruses (varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6) were also detected in some cases in both groups. Finally, we obtained clinical data from all positive patients and established a relationship with PCR results.

Meningoencefalitis por virus de la familia Herpesviridae en Cuba. Período 1996-1998. / [Meningoencephalitis caused by Herpesviridae family virus in Cuba. 1996-1998]

Herpesvirus isolation from cerebrospinal fluid (CSF) has proven not to be very useful because of its low sensitivity; that is the reason why PCR has considerably improved the diagnosis of several viral infections of the central nervous system. We studied, by means of PCR, 253 CSF received in our laboratory from 1996 to 1998, which had a suspected diagnosis of herpesviral-associated neurologic disease (encephalitits or meningoencephalitis). Of these, 182 were from HIV seropositive patients and the other 71 were from HIV seronegative. The percentage of all positive cases was 22.5

in AIDS patients and CMV was the virus most frequently detected (13.7

). We detected a percentage of 29.5 of positive cases in immunocompetent individuals, being HSV the virus most frequently found. Other herpesviruses (varicella-zoster virus, Epstein-Barr virus, human herpesvirus 6) were also detected in some cases in both groups. Finally, we obtained clinical data from all positive patients and established a relationship with PCR results.

[Prevalence of antibodies against herpes simplex virus, Epstein-Barr virus and cytomegalovirus in a group of patients after hemodialysis]. / Prevalencia de anticuerpos contra virus herpes simple, virus Epstein-Barr y citomegalovirus en un grupo de pacientes con hemodiálisis realizada.

Infections from herpes virus play a key role in post-transplantation pathology, so it is indispensable to characterize the group of would-be renal transplant recipients. IgG antibody titers were detected by indirect immunofluorescence to Herpes simplex virus, Epstein Barr virus (EBV) and cytomegalovirus (CMV) in 100 patients treated with hemodialysis in the Nephrology Institute. The prevalence of antibodies to cytomegalovirus and to Herpes simplex and Epstein Barr viruses was 100% and 95% respectively. Average geometric titers (AGT) of antibodies to CMV were significantly higher. There was no correlation between AGT and sex, age, time of disease remission, number of blood transfusions and number of transplants.

[Herpesvirus detection in immunocompromised patients with meningoencephalitis by the polymerase chain reaction technic]. / Detección de herpesvirus en pacientes inmunocomprometidos con meningoencefalitis, mediante la técnica de reacción en cadena de la polimerasa.

The multiple polymerase chain reaction technique was used to detect in a single assay tube the presence of herpes simplex virus (HSV), Cytomegalovirus (CMV). Epstein Barr virus (EBV), varicella zoster virus (VZV), and/or human herpes virus-6 (HHV6). 50 cerebrospinal fluids from patients with AIDS and clinically suspected to have meningoencephalitis due to HSV were received and analyzed at the laboratories of Sexually Transmitted Diseases of the Virology Department of the "Pedro Kourí" Tropical Medicine Institute from 1993 to 1996. 4 of them were positive to HSV, 3 to CMV, 2 to VZV, and 1 to HHV6 for a positivity of 20%. The results of the PCR were correlated to the clinical findings presented by the patients at the time of the lumbar puncture. The introduction of this technique in the laboratory allows to have an easy, fast and useful tool for the diagnosis of meningoencephalitis due to herpesvirus.

[Follow-up serological study of herpes simplex and cytomegalovirus in Cuban patients infected with human immunodeficiency virus (HIV)]. / Estudio serológico evolutivo a herpes simple y citomegalovirus en pacientes cubanos infectados con el virus de la inmunodeficiencia humana (VIH).

The evolution serological response against the herpes simplex virus and citomegalovirus in HIV infected patients grouped into different stages of the disease was studied. Fluctuations in the TPG of antibodies were observed in these values in a cyclical way through time. There was a greater significant difference among the TPG of antibodies against HSV in the group of asymptomatic patients compared with AIDS patients and with those who died. There is a marked decrease in the TPG of antibodies against HSV and CMV approximately one year before the death of patients.

[Serologic response to some herpesviruses in a group of HIV infected patients]. / Estudio de la respuesta serológica a algunos herpesvirus en un grupo de pacientes infectados con el VIH.

The titres of antibodies to the herpes simplex virus, cytomegalovirus, and the antigen of viral envelope of Epstein-Barr virus were determined in serum samples from patients infected by the human immunodeficiency virus (HIV) who belonged to groups II (n = 26) and IV (n = 32) according to the present Classification System of HIV infection, as well as in a group of blood donors (n = 52). The geometrical average titres obtained in the different agents were very high in patients having HIV infection with respect to the geometrical average titres observed in blood donors. Patients from group II had titres to the antigen of viral envelope of the Epstein-Barr virus significantly higher than blood donors. Levels of antibodies to these agents in patients from group IV were higher than levels obtained in blood donors. Differences regarding levels of the antigen of viral envelope of the Epstein-Barr virus were highly significant. These results suggest the possible relevance of the Epstein-Barr virus infection in the natural history of AIDS.