Polyclonal Pattern Research Articles

Acute hemorrhagic leukoencephalopathy associated with influenza A (H1N1) virus

Since the declaration of the novel influenza A (H1N1) pandemic by the World Health Organization in June 2009, several neurologic complications have been reported including encephalitis, encephalopathy, seizures, and neuropsychiatric perturbations [1, 2]. Most of the cases concerned children [1] and had a benign course with early and appropriate management. When performed, brain magnetic resonance imaging (MRI) was normal. We report the first case of H1N1 virus related to acute hemorrhagic leukoencephalopathy. A 56-year-old Caucasian woman without past medical history consulted her general practitioner 24 h after the onset of an influenza syndrome. She received imipenem per os 2 g per day for 10 days. On the fifth day, the patient was referred for permanent dyspnea, headache, and 38.5 C fever. No cognitive changes were observed. Pulmonary auscultation found crackles. A chest X-ray showed features of acute respiratory distress syndrome with bilateral infiltrative opacities. At admission, H1N1 was isolated from nasopharyngeal specimens by reverse-transcription polymerase chain reaction (RT-PCR) [3]. The virus could not be isolated by cell culture for sequencing. One hundred fifty milligrams oseltamivir and 40 mg methylprednisolone intravenous per day were administered over 5 days while imipenem was continued. On the sixth day, massive oxygen desaturation occurred despite noninvasive oxygenotherapy. The patient became comatose. She was transferred to the intensive care unit. Mechanical ventilation was then performed after intubation. Brain MRI with T2-, T2*-, and T1-weighted sequences with and without gadolinium infusion showed extensive bilateral hemispheric leukoencephalopathy predominating in the subcortical regions and extending in the right striatum. Several microbleeds were present in the same territories (Fig. 1a–g). MRI angiography was normal. Electroencephalography recorded bilateral slow waves. Cerebrospinal fluid (CSF) was xanthochromic and contained 0.51 g/L protein in a polyclonal pattern with 90 red blood cells and 8 lymphocytes/ mm. PCR was negative for cytomegalovirus, herpes simplex, Epstein–Barr, and H1N1 viruses. Specific antiH1N1 antibody titer determined by hemagglutination inhibition against the A/California/7/2009 strain was 10 in CSF and more than 1,280 in serum. Culture for bacteria and mycosis remained sterile. Neither antinuclear nor onconeural antibodies were noted. On the tenth day, the patient awoke but was deeply confused. The Mini-Mental State Examination (MMSE) score was 19/30 with deficit in time and space orientation and calculation errors. Neurological examination found a left pyramidal syndrome with 4/5 Medical Research Council score on limb muscle testing. Diagnosis of acute partially hemorrhagic leukoencephalopathy linked to the H1N1 virus was reached, and steroid therapy started with methylprednisolone 500 mg daily for 3 days. On the 40th day, muscle strength was normal. The MMSE was 29/30. An extensive neuropsychological expertise showed minor abnormalities with normal memory, verbal comprehension, and executive F. A. Cisse J.-C. Antoine G. Jousserand J.-P. Camdessanche (&) Department of Neurology, University Hospital, 42055 Saint-Etienne Cedex 02, France e-mail: j.philippe.camdessanche@chu-st-etienne.fr

Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis

Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma.IntroductionMalignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population [1]. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year [2]. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation [3,4]. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis [5,6].GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost [7]. Therefore, rapid and more cost effective technique are being sought.LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye [8,9]. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product [10,11]. Therefore, current investigations using melting curve analysis are being developed [12,13].In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.

Stable Marking and Transgene Expression Without Progression to Monoclonality in Canine Long-Term Hematopoietic Repopulating Cells Transduced with Lentiviral Vectors

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients.

Analysis of the TCR β-chain gene rearrangement and CDR3 repertoire in patients with breast cancer.

Objective To analyze the clonal gene rearrangement and complementarity determining region 3 (CDR3) repertoire of TCR β-chain in fine needle aspiration biopsy (FNAB) specimens of lymph node metastasis in patients with breast cancer. Methods The TCR CDR3 region genes of 24 TCR Vβ subfamilies were amplified by utilizing RT-PCR technology, and the CDR3 lengths of TCR β-chain were analyzed with gene scan technology for 2 cases with lymph node reactive hyperplasia and 3 patients with lymph node metastasis of breast cancer. The clonality of T cells presumed by spectra typing was further confirmed by CDR3 sequencing. Results TCR β-chain presented specific repertoire skewing in metastatic lymph node,and only 3-5 TCR Vβ subfamily of T cells were identified, respectively. Clonal expanded T cells, including oligoclonal, polyclonal patterns, in one or more Vβ subfamilies were found in all cases. The oligoclonal expanded T cells had different CDR3 amino acid sequences. Conclusion There are characteristic T cells cloning proliferation and selected usage of TCR Vβ subfamily T cells could be found in metastatic lymph node.The sequences of CDR3 in different TCR clone proliferation are mostly different. Key words: Breast neoplasms; Gene rearrangement; Complementarity determining regions; Gene scan

Gingival plasma cell granuloma

Plasma cell granuloma is a rare reactive lesion composed of polyclonal plasma cells. It manifests primarily in the lungs, but may occur in various other anatomic locations like the oral cavity. Intraoral plasma cell granulomas involving the tongue, lip, oral mucosa and gingiva have been reported in the past. This case presents a 54-year-old female with chronic periodontitis and mandibular anterior gingival overgrowth treated by Phase I therapy (scaling and root planing) and excisional biopsy. Histological examination revealed inflammatory cell infiltrate containing sheets of plasma cells. Immunohistochemistry for kappa and lambda light chains showed a polyclonal staining pattern confirming a diagnosis of plasma cell granuloma. This case highlights the need to biopsy for unusual lesions to rule out potential neoplasms.

Monoclonal Gammopathy of Undetermined Significance Disguised as Chronic Neutrophilic Leukemia

We encountered a 60-year-old woman with a medical history of diabetes mellitus, osteoporosis, peripheral vascular disease, and hypertension who had earlier presented at an outside facility with knee pain, which led to a finding of elevated neutrophil count of 35×109/L. Because she was otherwise asymptomatic but continued showing elevated neutrophil levels, she sought a second opinion at our facility. Serum protein immunoelectrophoresis with immunofixation revealed an immunoglobulin A (IgA)-κ monoclonal gammopathy concentration of 1305 mg/dL (normal 80–350 mg/dL) but relatively normal concentrations of IgG of 840 mg/dL (620–1400 mg/dL) and IgM of 36 mg/dL (45–250 mg/dL). Using clonal analysis, we found a polyclonal expression pattern in all cell types analyzed. Comprehensive work-up for multiple myeloma and infectious etiology of neutrophilia was negative. We concluded that our patient’s neutrophilia may have been due to the underlying monoclonal gammopathy. This is the first case in the literature of a patient with monoclonal gammopathy of undetermined significance presenting with chronic neutrophilia, mimicking chronic neutrophilic leukemia (CNL). Patients with CNL have a poor prognosis; therefore, it is important to distinguish diagnostically between CNL and reactive neutrophilia.

Monoclonal Gammopathy of Undetermined Significance (MGUS) Does Not Affect Outcomes in Patients Undergoing Solid Transplants.

Abstract 2834Poster Board II-810 Background:Monoclonal gammopathy of undetermined significance (MGUS) results arises from a clonal proliferation of differentiated plasma cells and is a necessary precursor to development of multiple myeloma and some other B cell malignancies. Despite a high incidence in the general population (1-2% of adults in the 6th and 7th decades) and increased relative risk for later malignancy, there are few reports about the clinical course and prognosis of MGUS in long-term immunosuppressed patients. We aimed to evaluate the significance of MGUS in patients undergoing solid transplants in terms of clinical outcomes and rate of transformation to secondary B cell malignancy. Material and methods:We reviewed the medical records of 1493 patients on which kidney (living donor related, living donor-unrelated and deceased donor), liver and pancreatic transplants were performed at Mayo Clinic Arizona, between January 1, 1999 and July 31, 2009 to determine the incidence of MGUS and to further evaluate long-term follow up. At MCA patients are routinely screened using SPEP and for those where an M-spike is detected or suspected immunofixation is required for confirmation. MGUS was defined as previously reported. Patients who had anemia unrelated to their PCD were included. Statistical Methods:Patients were observed until death or, for those still alive, until July 2009. The major goal of the study was to assess the impact of MGUS on transplant outcomes and progression to active myeloma or related disorders. Patients who did not experience progression were censored at the time of last follow-up. Nominal variables were compared using Fisher's exact test. A two-tailed P value less than .05 was considered significant. ResultsIn this series, SPEP was performed in 1199 out of 1593 cases (75%) before transplantation. The most common abnormalities detected by SPEP included a normal pattern (70%), polyclonal pattern (16.8%), hypoalbuminemia (8%), hypogammaglobulinemia (2.4%) and the presence of M spike (2.8%). Immunofixation revealed the presence of a monoclonal immunoglobulin in 34 patients: IgG 30 cases (88%), IgM in 2 (6%) and biclonal disease in 2 (6%). The median age at diagnosis of MGUS was 60 years (range, 46 to 84 years), and included 63.7% of males. Clinical characteristics of the MGUS cases are seen in Table 1. Two MGUS patients were diagnosed only after the transplant was performed (338 and 1082 days respectively). There was no association between post-transplant lymphoproliferative (PTLPD) and MGUS and even when some MGUS patients died (7/34, 20%) death was not associated with progression of the monoclonal clone or development of a PTLPD or other malignancy. MGUS does not impact transplant outcomes. PTLPD disorders developed in 6 cases and none was associated with a MGUS. After a median of 88 months no MGUS patient developed transformation to active myeloma or a related disorder. Overall survival after transplantation was not significantly different among MGUS and Non- MGUS cases (6.48 vs 8.36 years p>0.05). DiscussionDespite the frequency of MGUS in the general population there are few studies which have assessed MGUS in solid organ transplant patients. An increased incidence of monoclonal gammopathy in patients with autoimmune disease and in older non-transplant populations suggests that impaired immunity might lead to development of MGUS and later monoclonal B-cell malignancies. Interestingly, we identified two cases where MGUS appeared after transplant. In this series, we report on the incidence and follow/up of MGUS in patients screened before transplantation. The primary outcome for transplant recipients is unaffected by the presence of MGUS and incidence is in accordance with the expected frequency (2.8%). Patients with MGUS do not appear to be at risk for progression due to chronic immunosuppression. Testing for MGUS before transplantation is not cost-efficient and therefore would not be recommended. To our knowledge this is the largest series of MGUS and transplant outcome yet reported.Table 1Clinical characteristics among MGUS casesCharacteristicMedian and RangeN=34Age60 (46-84)GenderMale 25Female 9IgG30IgM2Biclonal2Kappa24Lambda9Kappa and lambda1BMPC (17)Median 3 (0-7%)M spikeMedian 0.4 (0.2-2.3)Evolving type0/34Progression to active myeloma0/34Abb: BMPC: Bone marrow plasma cell number Disclosures:Stewart:Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy; Takeda, Millenium: Research Funding; Genzyme, Celgene, Millenium, Proteolix: Honoraria.

The Expression Pattern of TCR Vγ I∼III Subfamilies in GVHD Patients After Allogenic Hematopoietic Stem Cell Transplantation.

Abstract 2219Poster Board II-196 ObjectiveThe γΔ+ T cells can act as a vast functional setting of immune defense against pathogenic invaders in chronic inflammatory reactions, as well as in modulating systemic and organ-specific autoimmune diseases. Recently, it was reported that γΔ+ T cells might play an important role in regulation of graft versus host disease (GVHD), mediation of graft versus leukemia effect, anti-virus infection, anti-cancer effect and so on. But the frequency of T cell receptor (TCR) Vγ repertoire and the expression patterns of TCR Vγ subfamilies in GVHD remain unknown. To further study on the characteristics of TCR Vγ subfamilies in GVHD, we investigated the frequency of TCR Vγ repertoire and the expression of TCR Vγ I∼III subfamilies genes in the patients with GVHD after allogenic hematopoietic stem cell transplantation (allo-HSCT). MethodsThe expression and cloanlity analysis of TCR Vγ repertoire was detected in peripheral blood mononuclear cells (PBMNCs) from 25 patients with GVHD by RT-PCR and genescan technique. The TCR Vγ I∼III genes expression levels of PBMNCs from patients with GVHD were detected using the real-time fluorescence quantitative polymerase chain reaction with SYBR Green I technique and relative quantification method. Twelve healthy individuals served as normal controls. ResultsThe RT-PCR results showed that in GVHD patients, the expression frequency of TCR Vγ I was 88.0% (22/25), TCR Vγ II was 72.0% (18/25), and TCR Vγ III was 96.0% (24/25). All of the three TCR Vγ subfamilies could be detected in PBMNCs from the normal controls. TCR Vγ repertoires with polyclonal pattern were identified in normal controls. However, the skew expression pattern of TCR Vγ repertoire could be detected in patients with GVHD even more than 4 year after allo-HSCT. Oligoclonal or monoclonal expanded T cells were identified in TCR Vγ I∼III subfamilies in GVHD patients. The expression level of TCR Vγ II gene in the PBMNCs from patients with GVHD was significant lower than the normal controls (P<0.05). The pattern of TCR Vγ gene expression levels in GVHD was TCR Vγ I> TCR Vγ III> TCR Vγ II, however, the expression pattern in normal controls was TCR Vγ II> TCR Vγ I > TCR Vγ III. ConclusionsThe expression patterns of TCR Vγ I∼III subfamilies were changed in GVHD. The low expression of TCR Vγ II subfamily might be related with the pathogenesis of GVHD. Disclosures:No relevant conflicts of interest to declare.

Extranodal Rosai-Dorfman disease of the kidney

Extranodal Rosai-Dorfman disease of the kidney

Analysis of T Cell Cloanlity of Ph+ Acute Lymphoblastic Leukemia with Chronic Gvhd in Continuous Remission after Allogeneic Hematopoietic Stem Cell Transplantation

Chronic graft-versus-host disease (cGVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, recent study suggested that the presence of cGVHD might reduce the risk of relapse in Ph+ acute lymphoblastic leukemia (ALL) after allo-HSCT. GVHD is mediated by proliferation of special T-cell clones, we analyzed the T cell receptor (TCR) Vβ repertoire and cloanlity in Ph+ ALL patients undergone cGVHD after allo-HSCT in order to find the special T-cell clones associated with continuous remission.Allogeneic transplantations were performed in the first complete remission (CR) (6 ALLP190+ patients). The numbers of BCR-ABL copies in peripheral blood samples were assessed with the real-time quantitative RT-PCR (RQ-PCR) test at diagnosis, and every month after HSCT. The quantity of BCR-ABL transcript was normalized for normal ABL expression and the result was expressed as the ratio of BCR-ABL copies to ABL copies. With monitoring BCR-ABL copies of 6 BCR-ABLP190+ ALL patients after HSCT (the mean ratio of BCR-ABLP190/ABL at diagnose was 0.41 ± 0.39%), we found that 4 patients with extensive cutancous cGVHD had no evidence of leukemia recurrence, and the BCRABL transcripts of patients with cGVHD were remaining 0 copy, while 2 patients without cGVHD relapsed (the mean ratio of BCR-ABLP190/ABL was 7.56 ± 2.49%) and even dead within six months of HSCT.The expression and cloanlity analysis of TCR Vβ repertoire was detected in peripheral blood samples from patients with cGVHD by RT-PCR and genescan technique. TCR Vβ repertoires with polyclonal pattern were identified in normal controls and donor groups. However, the skew expression pattern of TCR Vβ repertoire could be detected in patients with cGVHD even more than 1 year after allo-HSCT. Among the 24 Vβ subfamilies, there were only 11~17 Vβ subfamilies expressed in cGVHD patients. Oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 1, 2, 3, 7, 8, 12, 14, 16~20, 22 and 23 subfamilies in 4 ALL patients (BCR-ABLP190 remission) with cGVHD. As a contrast, oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 2, 7, 8, 14, 17, 20, and 22 subfamilies in 2 chronic myeloid leukemia patients (BCR-ABLP210 remission) with cGVHD.In conclusion, the predominant usage and clonal expansion of TCR Vβ subfamily T cells could be found in Ph+ ALL patients with cGVHD and these groups of specific T-cell clones (Vβ 1, 3, 12, 16, 18, 19 and 23) were potentially associated with cGVHD in ALL and might be responsible for maintaining CR in Ph+ ALL patients after allo-HSCT. Research Funding.

X-Linked Clonality Analysis of Human Embryonic Stem Cells ( hESC ) Before and after induction of Hematopoietic Differentiation

Human ES cell lines represent an invaluable tool of research for the study of normal and pathological development. In this study we asked whether the human ES cell lines currently used for research purposes are derived from single outgrowths using X-linked clonality analysis. Amongst the cell lines tested, the female H9 cell line was informative at the Humara (human androgen receptor) locus by the presence of two CAG repeats allowing distinction of paternal (Xp) and maternal (Xm) X chromosomes. Clonal analysis was performed upon Hpa II digestion of DNA purified from total H9 cells (passages 27–61), single H9 cell clones generated from single cell cloning, single embryoid bodies (EB) (day 4, day 14) as well as from single hematopoietic progenitors. The same analysis was performed after induction of hematopoietic differentiation in liquid cultures in the presence of hematopoietic permissive conditions (co-culture in the presence of OP-9 cell line). Clonality was evaluated by calculating the Relative Corrected Index (RCI) which is the ratio of the intensity of peaks generated by Hpa II-digested and undigested DNA. In several experiments, all passages of H9 cell lines examined ( p27, p28, p31, p61) were found to exhibit a monoclonal pattern with disappearance of the same allele A upon Hpa II digestion (RCI > 10). 13 clonal cultures of H9 obtained by single cell cloning exhibited also the same clonal pattern with digestion of the same allele and RCI values between 6–14. Similarly, DNA purified from EB's at different stages was found to be clonal. In DNA obtained from 75 individual hematopoietic progenitors (CFU-GM, CFU-G), clonal analysis revealed the same clonal pattern in each, with disappearance of the same allele A. However, clonal analysis performed in 10 hematopoietic colonies, revealed unexpectedly, an equal digestion of Humara alleles with RCI < 2 (polyclonal pattern). Finally, in two experiments, hematopoietic cells recovered from liquid cultures exhibited an absolute clonal pattern (Total disappearance of the same allele A). Interestingly, quantification of X-Inactivation Specific Transcript (XIST) RNA in H9 and day14 EB's by RT-PCR did not show any evidence of XIST expression both before and after differentiation. Altogether, our data suggest that XCI has already been initiated in undifferentiated H9 cells and this inactivation is maintained, at least in the Humara locus in the absence of XIST. The monoclonal pattern of the cell line can be due to the fact that H9 ES cell line might have arisen from a limited number of stem cells having undergone XCI at a very early stage, with maintenance of the methylation status at this locus during mesodermal and hematopoietic commitment. Conversely, hematopoietic differentiation might occur from several mesodermal progenitors having inactivated the same X chromosome in H9 at the Humara locus. Despite the fact that hematopoietic progenitors and differentiated hematopoietic cells originating from H9 exhibited also a clonal pattern, a partial reactivation of the inactive X chromosome during hematopoietic differentiation could occur, and may explain the “polyclonal” pattern obtained in 10 CFU-GM analyzed. Overall, X-linked clonality testing could be an important issue before considering the potential clinical applications, in all female hES cell lines.

The Frequency and Clonality of γδ+T Cells, distribution of TRGV and TRDV Repertoire in Cord Blood T Cells

Our previous study has showed the feature of distribution and clonality of T-cell receptor alpha variable region (TRAV) and TRBV subfamily T cells. Recently, data indicated the γδ+T cells may play an important role in mediated graft versus leukemia effect after stem cells transplantation and anti-cancer response. In order to farther characterize the repertoire of CB T-cells, the frequency of αβ+ and γδ+T cells were detected in cord blood (CB) by FACS, the CDR3 size of 4 TRGV and 8 TRDV subfamily genes were analyzed in mononuclear cells (MCs) from 16 cord blood samples, using RT-PCR and genescan technique. Ten healthy individuals served as control. To determine the expression level of TRGV subfamily genes, quantitative analysis of TRGV I–III subfamilies was performed by real-time PCR. Low percentage of CD3+TCRγδ+ cells (1.33±0.58%) was observed in CB. The frequency expression was found in TRGVI (93.75%) and TRGVII (81.25%) in CBMCs, whereas the TRGVIII was detected in 9 out of 16 samples (56.25%). In TRDV subfamilies, the mean value of the number of expressed subfamilies in CBMCs is higher than that from adult peripheral blood (PB) group. The most frequently expressed in CB were TRDV1 (100%), TRDV2(93.75), TRDV8 (93.75%) and TRDV3 (81.25%). The frequency of TRDV5 and TRDV8 in CBMCs were significant higher that that from PBMC. The most PCR products of TRGV and TRDV subfamilies from 10 cord blood displayed polyclonal rearrangement pattern, whereas one or two PCR products from 6 CB samples displayed oligoclonality or biclonality. In contrast, PCR products from 9 of 10 adult healthy controls contained at least an oligoclonal peak in different TRGV or TRDV subfamilies respectively. In the present study, all PCR products form TRGVIV were showed monoclonality, the direct sequencing results showed that the V region of TRGVIV joint direct to C region, indicating that there is no rearrangement in TRGVIV and it is a pseudogene. The sequence data have been submitted to genebank and the Accession Number is EU395807. The pattern of TRGV subfamily expression level in CBMCs was TRGVI>TRGVIII>TRGVII, in contrast, TRGVII>TRGVI>TRGVII was found in PBMCs. In conclusion, our results indicate polyclonal and more diverse TRDV segment usage in CB γδ+ T-cells. The pattern of TRGV expression levels was different between T cells from PB and CB.

TRGV and TRDV repertoire distribution and clonality of T cells from umbilical cord blood

Umbilical cord blood (CB) has been used as a valuable source of hematopoietic stem cells for allogeneic transplantation, specific CTL response and immunotherapy for decades. We previously analyzed the distribution and clonality of T-cell receptor alpha and beta variable region ( TRAV) and ( TRBV) of the subfamily T cell receptors in T cells from umbilical cord blood. Recent data indicated that γδ + T cells may play an important role in mediating the graft versus leukemia effect after stem cells transplantation and in anti-cancer response. In order to further characterize the repertoire of CB T-cells, the frequency of αβ + and γδ + T cells were examined in CB by FACS. The CDR3 size of 4 TRGV and 8 TRDV subfamily genes were analyzed in mononuclear cells (MCs) from 16 CB samples, using RT-PCR and genescan technique. To determine the expression level of TRGV subfamily genes, we performed quantitative analysis of TRGVI–III subfamilies by real-time PCR. Low percentage of CD3 +TCRγδ + cells was observed in CB. The frequency of expression in TRGVI, TRGVII and TRGVIII in CBMCs was 93.75%, 81.25% and 56.25%, respectively. The mean value of the number of expressed TRDV subfamilies in CBMCs is higher than that from adult peripheral blood (PB) group. The frequently expressed members in CB were TRDV1 (100%), TRDV2 (93.75%), TRDV8 (93.75%) and TRDV3 (81.25%), respectively. The frequencies of TRDV5 and TRDV8 in CBMCs were significantly higher than those from PBMCs. Most of the PCR products of TRGV and TRDV subfamilies from 10 CB samples displayed polyclonal rearrangement pattern, whereas one or two PCR products from 6 CB samples showed oligoclonality or biclonality. In contrast, PCR products from 9 of 10 adult healthy controls contained at least an oligoclonal peak in different TRGV or TRDV subfamilies respectively. The pattern of TRGV subfamily expression level in CBMCs was TRGVI > TRGVIII > TRGVII, and in contrast, TRGVII > TRGVI > TRGVIII was found in PBMCs. In conclusion, our results indicate polyclonal and more diverse TRDV segment usage in CB γδ + T-cells. The pattern of TRGV expression levels in CB T cells was found to be quite different from the one in PB T cells. These findings are apparently the first report regarding the repression pattern of TRGV repertoire in CB. It also provides a detailed profile of the global TRGV and TRDV repertoire and TRGVI-III expression levels in cord blood T cells in Chinese subjects. The biological significance of the differences observed between CB and PB is at present obscure. However, this study will definitively contribute to understand the cellular immune features better and to exploit more efficiently the therapeutic potentials of CB.

Soft tissue non-Hodgkin lymphoma of shoulder in a HIV patient: a report of a case and review of the literature

BackgroundThe risk of developing lymphoma is greatly increased in HIV infection. Musculoskeletal manifestations of the human immunodeficiency virus (HIV) are common and are sometimes the initial presentation of the disease. Muscle, bone, and joints are involved by septic arthritis, myopathies and neoplasms. HIV-related neoplastic processes that affect the musculoskeletal system include Kaposi's sarcoma and non-Hodgkin's lymphoma, the latter being mainly localized at lower extremities, spine and skull.Case presentationThe Authors report a case of a 34 year-old lady. In December 2003 the patient noted a painless mass on her right shoulder whose size increased progressively. In March 2004 she was diagnosed HIV positive and contemporary got pregnant. The patient decided to continue her pregnancy and to not undergo any diagnostic procedure and treatment. At the end of August she underwent a surgical ablation of the lesion that revealed a lesion of 7 cm × 7 cm × 3,3 cm. The histology showed B-cells expressing CD20, PAX-5, CD10, BCL-6 and MUM-1 with 70% Ki67 positive nuclei. The lesion was also negative for EBV infection and showed a monoclonal rearrangement of IgH chain and a polyclonal pattern for TCR gamma and beta. A final diagnosis of diffuse large B-cell lymphoma was made. The patient underwent postoperative chemotherapy. At four-years follow up the patient is symptom free and no local nor systemic recurrence of pathology has been noted on MRI control. HIV infection is still under control.ConclusionIn this report, we present a case of diffuse large B-cell lymphoma localized in the soft tissue of the shoulder in a HIV infected patient. Authors want to underline this case for the rare position, the big size and the association with HIV infection.

Pseudoglandular Hepatoid Differentiation in Endometrioid Carcinoma of the Ovary Simulates Oxyphilic Cell Change

A 42-year-old patient had a stage III ovarian endometrioid adenocarcinoma with areas of hepatoid carcinoma (HC) of clear cell and eosinophilic pseudoglandular type that was difficult to differentiate from endometrioid carcinoma of oxyphilic type and sex cord-stromal tumor. Immunohistochemically, endometrioid adenocarcinoma was positive for CA125, estrogen and progesterone receptors, CAM5.2, cytokeratin (CK) 7 and 19, and vimentin. HC areas were positive for hep par1, polyclonal carcinoembryonic antigens, CD10, alpha-fetoprotein, epithelial membrane antigens, and antimitochondrial antibodies and shared with endometrioid carcinoma focal CK7, and constant positive CK19, CAM5.2, and progesterone receptors. In the differential diagnosis, a hepatic immunophenotype of oxyphilic, mitochondriae-rich areas (demonstrated by antimitochondrial antibodies) was identified by HC specific (hep par1) and characteristic markers: canalicular, cytoplasmic and membranous polyclonal carcinoembryonic antigens, CD10 patterns, and alpha-fetoprotein). Trabecular and clear vacuolated areas of HC resembled luteinized cells of sex cord-stromal ovarian tumors, but the membranous positivity to CAM5.2 supported epithelial (hepatoid) identity. The partial preservation of an endometrioid immunophenotype in HC (positive CK7 and 19 and progesterone receptors) would support an origin from endometrioid carcinoma.

Hyperplasia of Hair Follicles and Other Adnexal Structures in Cutaneous Lymphoproliferative Disorders

We studied 53 cutaneous lymphoproliferative disorders, all of which manifested hair follicle hyperplasia. There were 42 cases conforming to the description of pseudolymphomatous folliculitis (PLF) and 11 cases of authentic lymphomas including mycosis fungoides, CD30+ anaplastic large cell lymphoma, diffuse large B-cell lymphoma, B-cell small cell lymphoma/leukemia, and peripheral T-cell lymphoma, not otherwise specified. All patients with PLF clinically presented with a solitary nodule preferentially involving the face. Beside hair follicle hyperplasia, the typical features were a dense infiltrate of small well-differentiated lymphocytes, lymphoplasmacytoid cells, plasma cells, and epithelioid histiocytes forming tiny granulomas. Some unusual or worrisome features recognized included eccrine/apocrine duct hyperplasia, subcutis/muscle infiltration, lymphocyte "smudging," single file infiltration, and large atypical cells. Immunohistochemically, T-cell predominant cases dominated in the series. All 34 tested cases revealed a polyclonal pattern of kappa and lambda immunoglobulin (Ig) light chain expression. In 4 cases, scattered CD30+ cells were identified. Monoclonal rearrangements of T-cell receptor (TCR) and IgH genes were detected in 19 and 3 cases respectively, including 1 case with dual T-cell receptor/IgH rearrangement. Three of 30 tested cases proved positive for herpes simplex virus-1, whereas herpes simplex virus-2 always tested negative. Of 31 cases tested for Borrelia burgdorferi, 30 specimens were negative. In 9 cases, fluorescent in situ hybridization for t(11;18) and t(14;18) revealed none of the above translocations. The most common treatment modality was surgical removal. Forty patients with a mean follow-up of 3.7 years included 39 patients with no evidence of disease and 1 individual with local recurrence. The comparison of "clonal cases of PLF" and those with polyclonal population or in which clonality remained undetermined revealed no differences between the 2 groups in the clinical presentation, pathologic, and immunohistochemical features. We conclude that hyperplasia of hair follicles and other adnexa can be seen not only in the condition currently known as PLF, but also in genuine cutaneous lymphomas and may be just a happenstance secondary to a basic pathologic process.

Meningeal dissemination in primary CNS lymphoma

The impact of meningeal dissemination in primary CNS lymphoma (PCNSL) is debated, and the reported frequency varies. We prospectively evaluated the diagnostic value of PCR in comparison with CSF cytomorphology and MRI for diagnosing meningeal dissemination in PCNSL. We evaluated 282 patients from a multicenter therapy study for PCNSL for the presence of meningeal dissemination: 205 with CSF cytomorphology, 171 with PCR of the rearranged immunoglobulin heavy-chain genes in CSF, and 217 with cranial MRI. Meningeal dissemination was found in 33 of 205 patients (16%) by cytomorphology, in 19 of 171 (11%) patients evaluated by PCR, and in 8 of 217 patients (4%) by MRI. Considering either of these methods, the relative frequency of meningeal dissemination was 17.4% (49 of 282 patients). PCR was monoclonal in 6 of 19 (32%) samples with positive cytomorphology, 1 of 13 samples (8%) with suspicious cytology, and in 10 of 105 (10%) cytologically negative samples. In 11 samples with positive and 12 with suspicious cytology, PCR showed only a polyclonal pattern. The probability of meningeal dissemination detection was higher in cases with CSF pleocytosis (>5/microL) with an OR of 2.48 (95% CI 1.15-5.34, p = 0.018). CSF protein had no predictive value for meningeal dissemination detection. We found a low rate of meningeal dissemination in primary CNS lymphoma in this large prospective study. The rate of discordant PCR and cytomorphologic results was high. Thus, the methods should be regarded as complementary. CSF pleocytosis had predictive value for meningeal dissemination detection.

Disappearance of bone marrow B cell clonal expansion in patients with type II hepatitis C virus-related cryoglobulinemic glomerulonephritis after clinical efficient rituximab therapy

Preliminary data provided encouraging results on efficacy and safety of B cell depletion with rituximab in active glomerulonephritis in the course of mixed cryoglobulinemia (MC).1 2 B cell oligomonoclonal expansion...

Skew in T cell receptor usage with polyclonal expansion in lesions of oral lichen planus without hepatitis C virus infection

Oral lichen planus (OLP) is a refractory disorder of the oral mucosa. Its predominant symptoms are pain and haphalgesia that impair the quality of life of patients. OLP develops via a T cell-mediated immune process. Here, we examined the characteristics of the infiltrating T cells in terms of the T cell receptor (TCR) repertoires, T cell clonality, T cell phenotypes and cytokine production profiles. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of OLP biopsies from 12 patients. The cytokine expression profiles and T cell phenotypes were measured by real-time quantitative polymerase chain reaction. We observed that there were skewed TCR repertoires in the tissue samples (TCRVA8-1, VA22-1, VB2-1, VB3-1 and VB5-1) and PBMCs (TCRVA8-1, VB2-1, VB3-1 and VB5-1) from OLP patients. Furthermore, the CDR3 distributions in the skewed TCR subfamilies exhibited polyclonal patterns. We observed increases in CD4(+) T lymphocytes, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and human leucocyte antigen D-related in the OLP tissue specimens. Taken together, the present results suggest that T cells bearing these TCRs are involved in the pathogenesis of OLP, and that IL-5 and TNF-alpha may participate in its inflammatory process.

Polyclonal B-cell lymphocytosis with hairy cell appearance: hairy B-cell lymphoproliferative disorder

A 72-year-old woman was referred to our hospital for evaluation of leukocytosis revealed by a medical examination. Her physical examination demonstrated no splenomegaly and no palpable lymph nodes. Her white cell count was 10,900/microl with atypical lymphocytosis (84.5%). Her hemoglobin concentration was 10.4 g/dl, and platelet count 151,000/microl. On peripheral blood smears, the atypical lymphocytes had a hairy cell-like appearance, and phase-contrast microscopic and transmission electron microscopic findings revealed the lymphocytes had many long surface microvilli. Flowcytometric analysis of peripheral blood lymphocytes identified expanded B-lymphocytes as having the IgG+, CD5- CD10- CD11c+ CD19+ CD20+ CD23- CD25- and CD103- cell surface phenotype. Serum electrophoresis disclosed polyclonal elevation of IgG and IgM (2620 mg/dl and 840 mg/dl, respectively). No light-chain restriction and a polyclonal VH gene rearrangement pattern indicated the polyclonal proliferation of B cells. The patient was a nonsmoker and had HLA-DR4, as in previous reports which have suggested an association between hairy B-cell lymphoproliferative disorder (HBLD) and HLA-DR4. No chromosome 3 abnormality was observed. These findings were consistent with the characteristics of HBLD, but differed in some respects from those of persistent polyclonal B-cell lymphocytosis (PPBL). Therefore, we diagnosed this patient as having HBLD.