MICA4/HLA-DRB1*04/TNF1 haplotype is associated with mixed connective tissue disease in Swedish patients

In order to investigate major histocompatibility complex (MHC) class I chain-related gene A (MICA), tumor necrosis factor (TNFa), -308TNFA, and human leukocyte antigen (HLA-DR/DQ) polymorphisms in mixed connective tissue disease (MCTD), we analyzed 24 patients and 229 healthy controls from Sweden. MICA and TNFa typing was performed by polymerase chain reaction (PCR) and genotyping. HLA-DR and -DQ were genotyped using PCR-sequence specific primers (PCR-SSP) and PCR-sequence-specific oligonucleotide probe (PCR-SSOP), respectively. For analysis of -308TNFA polymorphisms we performed PCR with restriction endonuclease enzymes. We found that the MICA5.1-5.1 genotype was positively associated with MCTD. Shared epitope genes (DRB1*01 and DRB1*04) were also significantly positively associated with MCTD. Polymorphism of -308TNFA was not differently distributed in MCTD patients compared with controls. Furthermore, we demonstrated that frequencies of three estimated haplotypes were increased in MCTD patients compared with controls. Interestingly, the haplotype with MICA allele 4 together with DRB1*04 and TNF1 alleles gives the most specific pattern for MCTD patients compared with controls. Our study demonstrates a clear contribution of HLA loci in susceptibility to MCTD in the Swedish population. Susceptibility to MCTD may be linked to the MICA4/HLA-DRB1*04/TNF1 haplotype and MICA 5.1-5.1 genotype. Mixed connective tissue disease was also associated with shared epitope genes, which in RA has been associated with a more severe disease. Whether these genotypes affect the clinical phenotype of MCTD needs to be determined.     

Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults

It is well known that type 1 diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta 32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta 32 gene polymorphism and TIDM and we describe a new method for a simple and more precise genotyping of the CCR2 gene.     

Clinical features of double infection with tick-borne encephalitis and Lyme borreliosis transmitted by tick bite

Background

In Latvia and other endemic regions, a single tick bite has the potential to transmit both tick‐borne encephalitis (TBE) and Lyme borreliosis.

Objective

To analyse both the clinical features and differential diagnosis of combined tick‐borne infection with TBE and Lyme borreliosis, in 51 patients with serological evidence, of whom 69% had tick bites.

Results

Biphasic fever suggestive of TBE occurred in 55% of the patients. Meningitis occurred in 92%, with painful radicular symptoms in 39%. Muscle weakness occurred in 41%; in 29% the flaccid paralysis was compatible with TBE. Only two patients presented with the bulbar palsy typical of TBE. Typical Lyme borreliosis facial palsy occurred in three patients. Typical TBE oculomotor disturbances occurred in two. Other features typical of Lyme borreliosis detected in our patients were distal peripheral neuropathy (n = 4), arthralgia (n = 9), local erythema 1–12 days after tick bite (n = 7) and erythema chronicum migrans (n = 1). Echocardiogram abnormalities occurred in 15.

Conclusions

Patients with double infection with TBE and Lyme borreliosis fell into three main clinical groups: febrile illness, 3 (6%); meningitis, 15 (30%); central or peripheral neurological deficit (meningoencephalitis, meningomyelitis, meningoradiculitis and polyradiculoneuritis), 33 (65%). Systemic features pointing to Lyme borreliosis were found in 25 patients (49%); immunoglobulin (Ig)M antibodies to borreliosis were present in 18 of them. The clinical occurrence of both Lyme borreliosis and TBE vary after exposure to tick bite, and the neurological manifestations of each disorder vary widely, with considerable overlap. This observational study provides no evidence that co‐infection produces unusual manifestations due to unpredicted interaction between the two diseases. Patients with tick exposure presenting with acute neurological symptoms in areas endemic for both Lyme borreliosis and TBE should be investigated for both conditions. The threshold for simultaneous treatment of both conditions should be low, given the possibility of co‐occurrence and the difficulty in ascribing individual neurological manifestations to one condition or the other.The Baltic region is an endemic focus for both tick‐borne encephalitis (TBE) and Lyme borreliosis transmitted by ticks.^1,2,3,4 In Latvia, 7061 cases of TBE and 3566 cases of Lyme borreliosis were registered between 1994 and 2003, out of a population of 2.4 million. Both tick species present in Latvia, Ixodes ricinus and persulcatus, can transmit the encephalitis virus, the borreliosis spirochete and more rarely erlichiosis. A single tick bite has the potential to transmit both infections.⁵ Despite their different clinical courses, TBE and Lyme borreliosis have neurological features in common: lymphocytic meningitis, flaccid or spastic limb weakness and cranial nerve involvement. Thus, differentiating between these disorders is important, given different approaches to treatment.Of the two infections, only TBE runs a biphasic course with the initial prodomal period of influenza‐like symptoms usually developing 1–2 weeks after the tick bite. Hence, after an asymptomatic period lasting 2–10 days, about a third of infected patients enter a second phase with aseptic meningitis.² Subsequently, 2–10% in Western TBE subtype or 10–25% in Eastern TBE subtype develop encephalitis, myelitis or meningoencephalomyelitis typically manifesting as combinations of flaccid paresis of the limbs, usually arms and neck, bulbar dysfunction, disorientation, aphasia and spastic paresis.^1,2 A poliomyelitis‐like syndrome is described in central European TBE.⁶ Manifestations of TBE in the Baltic may be heterogeneous, given that infection with the Western, Far Eastern and Siberian subtypes all cause human infection in Latvia.⁷ Although severe manifestations usually subside after 3–6 weeks, the convalescence period of TBE may be very long, with nearly 40% having a postencephalitic syndrome at 4 years.⁸ The uptake of TBE vaccination is increasing in the Baltic region.Classical Lyme borreliosis differs considerably from TBE and produces local and generalised forms, systemic involvement, and development over several stages. Its acute and chronic courses pose problems of diagnosis and management.^1,9 Diagnosis of neuroborreliosis requires a definite or possible tick bite, erythema migrans or seropositivity, and typical peripheral or central nervous system involvement.¹⁰ In early neuroborreliosis (2–10 weeks after tick bite) the most common neurological abnormalities are meningitis, meningoradiculoneuritis and cranial neuritis, particularly facial palsy.^1,9,10,11 Progressive chronic encephalomyelitis, polyneuritis and cerebrovascular disorders are later manifestations of Lyme borreliosis, usually occurring months after the initial infection. Neurological features are noted in 10–12% of all patients with Lyme borreliosis in Europe¹ and in 10–15% of patients in Northern America.¹¹ Neurological manifestations in 330 European patients with Lyme borreliosis included radicular pain (70%), headache (18%), peripheral paresis (45%), central paresis (4%), sensory disturbances (44%) and facial palsy (39%).¹ Borrelia infection takes a subclinical or minimally symptomatic course in up to 80% of the population after tick bites.¹² Importantly, borreliosis is treatable with antibiotics.TBE infection can be proven by specific and sensitive ELISA detection of antibody in cerebrospinal fluid (CSF), or by detection of genome through polymerase chain reaction.¹³ Serum IgM antibodies can remain positive for ⩾10 months.^2,14 By contrast, serological tests for Lyme borreliosis infection are less sensitive and specific to variable onset and occurrence of specific IgM and IgG antibodies, with recognised persistent seronegatives; direct detection of a pathogen is rarely possible, and reliance must be placed on interpreting the laboratory investigations in the light of the clinical picture.^13,15,16 Demonstration of intrathecal antibody production provides a specific test,¹⁷ but is not sensitive in detecting all forms of neuroborreliosis.¹⁵ Despite their different clinical courses, TBE and Lyme borreliosis have neurological features in common: lymphocytic meningitis, flaccid or spastic limb weakness, and cranial nerve involvement. Pain, particularly in a radicular distribution, and sensory disturbance are regarded as features more typical of Lyme borreliosis than TBE.Only limited information on double infection with TBE and Lyme borreliosis is available. Single cases, small series or serologically defined series with limited clinical information are described from Germany, Slovenia, Central Russia and Finland.^{18,19,20,21,22,23,24} This retrospective clinical observational study analyses the clinical features and problems of differential diagnosis in patients with evidence of both TBE and Lyme borreliosis infection in Latvia.     

An outbreak of HBV and HCV infection in a paediatric oncology ward: epidemiological investigations and prevention of further spread

Hospital-acquired hepatitis B (HBV) and C virus (HCV) infections continue to occur despite increased awareness of this problem among the medical community. One hundred six patients were infected in a haematology oncology ward for children, over the time period 1996 to 2000. Serum samples from 45 such patients and 3 from infected medical personnel were used for nucleic acid amplification. HBV core, as well as HCV core and hypervariable region 1 (HVR1) nucleotide sequences, were analysed by phylogenetic tree analysis, in order to characterise the epidemiological pattern of viral transmission on the ward. Samples from 32 patients were positive for HBV-DNA or HCV-RNA by PCR. Ten patients were positive for both markers. Seventeen out of twenty-three HCV core gene sequences were found to be evolutionarily related and clustered separately from other local sequences in the phylogenetic tree, indicating nosocomial transmission. This was confirmed by analysis of HVR1 gene sequences. One nurse and one physician from the ward were HCV RNA positive, but their HCV sequences were not related evolutionarily to those of the patient cluster. Fifteen out of nineteen HBV core gene sequences were also clustered together and were positioned separately in the relevant tree. Epidemiological investigation excluded a common source infection and indicated that spread of infection was most likely due to inappropriate infection control measures on the ward. No obvious risk factors for transmission were identified during the retrospective survey in patients with related sequences, except use of multidose vials for saline and poor staff compliance with routine hand hygiene procedures. The preventive measures that were introduced reduced the incidence of infection significantly. No new cases of HBV infection and only three anti-HCV seroconversions occurred over a period of 19 months. The introduction and maintenance of strict prevention measures over a 2 year period, combined with HBV vaccination, reduced significantly the incidence of new HCV and HBV infections.     

Bone formation following implantation of bone biomaterials into extraction sites

BACKGROUND: Adequate bone volume is imperative for the osseointegration of endosseous implants, but postextraction resorption and remodeling may challenge implant placement. The use of bone biomaterials has been advocated to fill extraction sites and to enhance primary implant stability during osseointegration. The objective of the case series was to evaluate bone formation histologically and biomechanically in extraction sites following implantation of three commercially available bone biomaterials to compare their ability to allow guided bone regeneration. METHODS: Thirty-six periodontally involved teeth were extracted from eight healthy non-smoking subjects. At least two bone biomaterials, a synthetic sponge based on polylactic-polyglycolic acid technology (FIS), bovine porous bone mineral (BPBM), or a natural coral derivative physically and chemically transformed into a calcium carbonate ceramic (COR), and one non-grafted control were applied to the extraction sockets within each subject and were covered by an expanded polytetrafluoroethylene device. The devices were removed after 2 months, and trephine biopsies were obtained from each site 4 months later. At that time, endosseous implants were placed in 25 of the sites, and healing abutments were placed; measurements were taken 4 to 6 months later with an electronic mobility testing device. RESULTS: The percentage of residual biomaterial was 5.6% +/- 8.9% for FIS (P <0.001), 20.2% +/- 17.0% for BPBM (P <0.05), and 12.0% +/- 16.4% for COR (P <0.001). The amount of residual biomaterial after 6 months showed a significant relationship with the insertion torque measurements during the first third of implant insertion (P <0.05) and with values of the electronic mobility testing device at the abutment connection (P = 0.05). Histologically, new bone apposition was seen on BPBM particles. FIS sites showed similar ingrowth of blood vessels and osteocytes as empty controls. CONCLUSION: All sites revealed good primary stability at implant insertion and proper implant rigidity at abutment placement, indicating that early implant osseointegration was not influenced by the application of bone biomaterials used in this study.     

Performance comparison of two whole genome amplification techniques in frame of multifactor preimplantation genetic testing

Purpose

To compare multiple displacement amplification and OmniPlex whole genome amplification technique performance during array comparative genome hybridization (aCGH), Sanger sequencing, SNaPshot and fragment size analysis downstream applications in frame of multifactor embryo preimplantation genetic testing.

Methods

Preclinical workup included linked short tandem repeat (STR) marker selection and primer design for loci of interest. It was followed by a family haplotyping, after which an in vitro fertilization preimplantation genetic testing (IVF-PGT) cycle was carried out. A total of 62 embryos were retrieved from nine couples with a confirmed single gene disorder being transmitted in their family with various inheritance traits—autosomal dominant (genes—ACTA2, HTT, KRT14), autosomal recessive (genes—ALOX12B, TPP1, GLB1) and X-linked (genes—MTM1, DMD). Whole genome amplification (WGA) for the day 5 embryo trophectoderm single biopsies was carried out by multiple displacement amplification (MDA) or polymerase chain reaction (PCR)-based technology OmniPlex and was used for direct (Sanger sequencing, fragment size analysis, SNaPshot) and indirect mutation assessment (STR marker haplotyping), and embryo aneuploidy testing by array comparative genome hybridization (aCGH).

Results

Family haplotyping revealed informative/semi-informative microsatellite markers for all clinical cases for all types of inheritance. Indirect testing gave a persuasive conclusion for all embryos assessed, which was confirmed through direct testing. The overall allele dropout (ADO) rate was higher for PCR-based WGA, and MDA shows a better genomic recovery scale. Five euploid embryos were subjected to elective single embryo transfer (eSET), which resulted in four clinical pregnancies and birth of two healthy children, which proved free of disease causative variants running in the family postnataly.

Conclusions

A developed multifactor PGT protocol can be adapted and applied to virtually any genetic condition and is capable of improving single gene disorder preimplantation genetic testing in a patient-tailored manner thus increasing pregnancy rates, saving costs and increasing patient reliability.

     

Association between the transmembrane region polymorphism of MHC class I chain related gene-A and type 1 diabetes mellitus in Sweden

Major histocompatibility complex (MHC) class I chain related gene-A (MIC-A) is associated with type 1 diabetes mellitus (T1DM) in other populations. We tested the association of MIC-A gene polymorphism with T1DM in Swedish Caucasians; if it has an age-dependent association; and if the association has an effect on gender. We studied 635 T1DM patients and 503 matched controls in the age group of 0-35 years old. MIC-A5 was significantly increased in T1DM compared with controls (odds ratio [OR] =1.81, p(c) < 0.0005). Logistic regression analysis revealed MIC-A5 association was independent of HLA. MIC-A5 with DR4-DQ8 or MIC-A5 with DR3-DQ2 gave higher OR than the OR obtained with either of them alone (OR = 1.81, 7.1, and 3.6, respectively). MIC-A5 was positively (OR = 2.48, p(c) < 0.0005) and MIC-A6 negatively associated (OR = 0.61, p(c) = 0.035) with the disease in < or = 20 years of age. The negative association of MIC-A6 in young onset was confirmed by logistic regression analysis. MIC-A5 was associated with the disease in males (OR = 2.05, p(c) = 0.0005). MIC-A6 conferred protection (OR = 0.098, p(c) = 0.032) in females heterozygous for DR3/DR4. In conclusion, MIC-A5 is associated with T1DM; the association was higher in individuals < or = 20 years old; and negative association of MIC-A6 was stronger in younger onset patients than in older onset patients.     

Periodontal parameters around implants anchoring orthodontic appliances: a series of case reports

BACKGROUND: Implants replacing missing teeth provide advantages over clinical orthodontic treatment as compensation for reaction forces is no longer necessary and the lack of teeth is immediately resolved. METHODS: A total of 38 two-stage implants were inserted (16 in maxilla, 22 in mandible) in 10 partially edentulous patients with orthodontic problems. Osseointegration and marginal bone levels were assessed via intra-oral radiographs taken at the abutment stage and at the completion of the orthodontic treatment and also via probing depth, measurement of recession toward the implant/abutment (I/A) interface, and sulcus bleeding index, recorded after completion of orthodontic treatment. RESULTS: In the maxilla, the cumulative survival rate was 87.1% after 2 years; for the mandible, it remained 100%. Mean amount of bone loss was 1.6 mm for maxilla and 0.8 mm for mandible. No correlation could be found between directions of orthodontic forces and marginal bone loss. Mean percentage of bleeding sites was 38.5% and 25%, respectively, for implants in the maxilla and mandible. Attachment level was 1.2 mm (SD: 1.2) below I/A interface after completion of the orthodonic treatment. CONCLUSION: No significant marginal bone loss was present. Using implants during orthodontics can result in an easier and more predictable treatment.     

Tissue evacuated during joint replacement procedure as a source of mononuclear cells

Background

Different cell populations from bone marrow were used in various clinical trials for cardiac diseases during last decade. Four clinical studies are ongoing in our institution and enroll patients with cardiac diseases, coronary disease, type 2 diabetes, and osteoarthritis. The density gradient is used to separate bone marrow mononuclear cells. Joint replacement procedures were associated with significant loss of tissue. Usually, excess tissue as bone marrow, peripheral blood and fat are removed to clean operation site. The aim of this study is to prove whether removed tissue during joint replacement procedure can be considered as a significant source of mononuclear cells.

Methods

Excised tissue obtained during joint replacement procedure was collected by AutoLog system. Bone marrow tissue was collected by iliac crest puncture. Mononuclear cells from both sources were isolated by using Ficoll density gradient centrifugation. Flow cytometry was used to detect mononuclear cell, CD34+ population counts and cell viability. Tissue processing yields between the group of joint replacement and iliac crest puncture group were compared.

Results

Together, 34 bone marrow tissue processings were performed. On average, samples contained 46.31 ± 9.35 ml of bone marrow solution. Average cell yield in final product was 28.64 ± 9.35 × 10⁶ MNCs and 0.77 ± 1.51 × 10⁶ CD34+ population. In case of tissue removed during joint replacement nine processings were performed. On average samples contained 450 ± 157.69 ml of tissue solution. Average cell yield in final product was 76.67 ± 35.42 × 10⁶ MNCs and 1.33 ± 0.97 × 10⁶ CD34+ population.

Conclusions

Tissue processing analysis shows that tissue removed during joint replacement procedure can be assumed as a significant source of mononuclear cells. Methods used for bone marrow-derived mononuclear cell extraction can be applied to the excess tissue.