CXCL13 expression in Chlamydia trachomatis infection of the female reproductive tract.

Chlamydia trachomatis is the most common cause of acute salpingitis worldwide. The socioeconomic impact of sexually transmitted infections (STI) caused by C. trachomatis is considerable. The purpose of this study was to investigate secretion of a unique chemokine, CXCL13, during the inflammatory process in human fallopian tube tissue in response to infection with C. trachomatis. We employed two models for our experiments: archived fallopian tube paraffin sections from known cases of salpingitis of unknown etiology and human fallopian tube organ culture established from fresh fallopian tube biopsies subsequently infected in vitro with C. trachomatis serovar E. We used immunohistochemistry, microarray analysis and cytometric bead array to study these specimens. In both models, we found that the fallopian tissue infected with C. trachomatis expressed CXCL13 and other characteristics of tertiary lymphoid tissue. In addition, we found that CXCL13 was expressed in multiple cell types, including endothelial cells, demonstrating a mechanism for the lymphoid aggregation seen in fallopian tube tissue during salpingitis and infection with C. trachomatis.

DNA topoisomerase IIalpha in multiple myeloma: a marker of cell proliferation and not drug resistance.

DNA topoisomerase IIalpha (topo IIalpha) is the target for a number of antineoplastic agents. Down-regulation of this enzyme is one form of drug resistance. Topo IIalpha is also involved in DNA replication and transcription and serves as an indicator of proliferation rate in many human malignancies. This study examines whether topo IIalpha is one of the mechanisms of chemoresistance commonly observed in multiple myeloma (MM) or alternatively, whether topo IIalpha is associated with tumor cell proliferation. Bone marrow (BM) biopsy sections from 72 cases of MM, stratified according to proliferative activity (bromodeoxyuridine uptake), were immunostained for topo IIalpha. Immunoreactivity with an additional marker of drug resistance, glutathione-S-transferase pi, and the proliferation marker Ki-67 were also examined. Topo IIalpha was expressed in 26 (36%) cases and correlated strongly with proliferative activity (P <.001). A role for drug resistance could not be supported, given this strong relationship with proliferation and the finding that glutathione-S-transferase pi expression in 57 (78%) cases was independent of topo IIalpha immunoreactivity. Topo IIalpha was identified in 91 to 100% of highly proliferative tumors, as evaluated by bromodeoxyuridine uptake or Ki-67 reactivity, respectively. Proliferation also correlated with the histologic grade of the MM. Therefore, topo IIalpha immunoreactivity is primarily a marker of cell proliferation in MM and as such is likely to have prognostic significance. Highly proliferative tumors are most likely to be sensitive to chemotherapeutic protocols using anti-topo IIalpha agents.

Expression of a free gamma heavy chain in serum following autologous stem cell transplantation for IgG kappa multiple myeloma.

A 41-year-old male with IgG kappa multiple myeloma is described. He developed a free gamma heavy chain without an accompanying light chain following high-dose chemotherapy and autologous peripheral blood stem cell transplantation. The free gamma heavy chain was detected in serum and urine specimens 2 months after transplant, and eventually evolved into an IgG kappa monoclonal protein with electrophoretic properties similar to the original myeloma protein. Although the origin of the free gamma heavy chain remains uncertain, it was most likely related to the underlying plasma cell malignancy and, therefore, was an early sign of disease relapse.

Cyclin D1 and E2F-1 immunoreactivity in bone marrow biopsy specimens of multiple myeloma: relationship to proliferative activity, cytogenetic abnormalities and DNA ploidy.

Cyclin D1, encoded by the CCND1 gene, is immunohistochemically detectable in up to one-third of cases of multiple myeloma (MM). To examine the mechanism of cyclin D1 overexpression, we compared cyclin D1 immunoreactivity with the results of conventional cytogenetics to determine if the t(11;14)(q13;q32) or other abnormalities of 11q11-14 explained cyclin D1 overexpression. Karyotypic abnormalities were found in 45 out of 67 (67%) MM cases; the t(11;14) was present in seven cases (10%). Additional 11q11-14 abnormalities were not identified. The t(11;14) correlated with cyclin D1 upregulation in low to intermediately proliferative MM, but was not present in highly proliferative tumours (assessed using bromodeoxyuridine labelling index). Cyclin D1 indirectly activates the transcription factor E2F-1. In the bone marrow biopsy specimens of MM cases, E2F-1 was concurrently expressed with cyclin D1 (P = 0.001), indicating that cyclin D1 is functional. However, as neither E2F-1 nor cyclin D1 expression correlated with proliferative activity, the speculation that t(11;14) upregulates the CCND1 gene to induce higher proliferation and possibly more aggressive disease is not supported. We conclude that in low to intermediately proliferative MM cases, cyclin D1 is probably upregulated by t(11;14), but an alternative mechanism is more probable in highly proliferative MM.

Dendritic cells derived from multiple myeloma patients efficiently internalize different classes of myeloma protein.

Myeloma protein is a unique tumor antigen that can be used to devise tumor-specific vaccination strategies. As dendritic cells (DCs) are extremely potent at inducing T-cell responses, clinical protocols have been designed using myeloma protein-pulsed DCs to elicit anti-tumor cell responses in vivo. To optimize antigen pulsing of DCs, we investigated mechanisms of antigen uptake and evaluated various laboratory parameters including class of myeloma protein, antigen exposure time, and DC maturational stage.DCs were generated by culturing peripheral blood stem cells from myeloma patients in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Myeloma proteins were labeled with fluorescein isothiocyanate (FITC) and internalization of protein by DCs was measured by flow cytometry.IgG, IgA, and free-kappa light chain myeloma proteins were all rapidly internalized by DCs in a time-dependent fashion. Maturation of DCs with tumor necrosis factor-alpha (TNF-alpha) resulted in diminished uptake. Endocytosis of myeloma protein by DCs was primarily mediated by fluid-phase macropinocytosis based on morphology, nonsaturable uptake kinetics, and sensitivity to drugs that inhibit membrane ruffling. Pulse-chase experiments revealed that the majority of internalized myeloma protein disappeared within 4 hours but was retained in the presence of chloroquine, indicating antigen processing had occurred. Cultured DCs from myeloma patients are functional and can efficiently endocytose different classes of myeloma protein by the mechanism of macropinocytosis. This demonstrates the feasibility of using all classes of myeloma protein for producing DC vaccines, and defines culture conditions for optimizing antigen loading of DCs for induction of anti-myeloma responses.

A case of aggressive multiple myeloma with cleaved, multilobated, and monocytoid nuclei, and no serum monoclonal gammopathy.

Multiple myeloma is a B-cell malignancy characterized by proliferation of neoplastic plasma cells. A few cases have been reported identifying variant forms of neoplastic plasma cells with atypical nuclei that secrete myeloma protein. We report a highly unusual case of plasma cell myeloma that presented with cleaved, multilobated, and monocytoid nuclei, without detectable myeloma protein in the serum or urine. The bone marrow contained sheets of plasma cells exhibiting pleomorphic nuclei with cleaved, multilobated, and monocytoid features that were negative for myeloperoxidase and dual esterase. Flow cytometric analysis revealed CD38high/CD45low cells expressing cytoplasmic kappa light chain, without evidence of myeloid or lymphoid differentiation. Following chemotherapy, the patient developed secondary plasma cell leukemia. A high plasma cell labeling index was obtained from bone marrow and peripheral blood, indicating a poor prognosis. In addition to quantitative immunoglobulins, serum protein electrophoresis, and immunofixation electrophoresis of serum and urine, we recommend cytochemical and flow cytometric studies for evaluation of suspected plasma cell myeloma with atypical cellular features.

Dendritic cell biology and the application of dendritic cells to immunotherapy of multiple myeloma.

Dendritic cells (DCs) are extremely efficient antigen-presenting cells that are potent stimulators of both B and T cell immune responses. Although DCs are normally present in extremely small numbers in the circulation, recent advances in DC biology have made it possible to generate DCs in culture. DCs can be generated in vitro from various cellular sources including bone marrow, cord blood and peripheral blood. Although culture conditions are extremely diverse, the majority of protocols grow DCs in GM-CSF and either TNF-alpha and/or IL-4. The addition of other growth factors such as SCF and Flt-3 ligand can dramatically enhance DC recovery. It is important to appreciate that DC subsets have been identified. Thus, DC at different stages of maturation, based on phenotype and capacity to capture antigen, can be obtained depending on culture conditions. For clinical applications, DCs can be generated in serum-free media and cryopreserved for future clinical applications. The ability to obtain DCs in numbers suitable for manipulating immune responses has pushed DC-based immunotherapies into the spotlight for treatment of various malignancies, including multiple myeloma, a B cell malignancy that is presently incurable. Although high-dose chemotherapy and transplantation have improved complete remission rates and overall survival in myeloma, immunotherapeutic strategies are needed for the additional cytoreduction needed to achieve a cure. Because DCs specialize in antigen capture and are extremely potent at stimulating T cell responses, they are ideally suited for generating anti-myeloma T cell responses in vivo. Several studies have demonstrated that myeloma protein, also called idiotype (Id), is sufficiently immunogenic and can be used to generate in vivo T cell responses in myeloma patients. Clinical trials using Id-pulsed DCs as a vaccine to treat minimal residual disease or relapsed myeloma are currently underway. Feasibility studies indicate that antigen-pulsed autologous DCs can be used to elicit in vivo Id-specific T cell responses. Additional studies are needed to optimize current DC vaccination protocols and determine clinical benefits associated with this approach. It is hoped that, following conventional therapies, a combination of adoptive immunotherapeutic modalities such as DCs together with myeloma-specific T cells may lead to improved clinical responses in multiple myeloma, and ultimately lead to complete remission and cure.

Human follicular dendritic cells inhibit superantigen-induced T-cell proliferation by distinct mechanisms.

Follicular dendritic cells (FDCs) reside within germinal centers of secondary lymphoid tissue where they play a critical role in antigen-driven immune responses. FDCs express numerous adhesion molecules that facilitate cellular interactions with B and T cells within the germinal center microenvironment. Although human FDCs have been shown to influence B-cell development, very little is known about the ability of FDCs to regulate T-cell responses. To investigate this functional aspect of FDCs, highly enriched preparations were isolated by magnetic cell separation using the FDC-restricted monoclonal antibody HJ2. We found that isolated human FDCs inhibited proliferation of both autologous and allogeneic T cells, and were dependent on the number of FDCs present. Inhibition by FDCs was observed using two serologically distinct superantigens at multiple concentrations (Staphylococcus enterotoxin A and B). In contrast, B cells failed to inhibit, and often augmented superantigen-induced T-cell proliferation. Antibody-blocking studies showed that CD54 and CD106 were involved in the ability of FDC to inhibit T-cell proliferative responses. When FDCs and T cells were separated by a semipermeable membrane, the inhibitory effect was partially abrogated, demonstrating that in addition to cell-cell interactions, a soluble factor(s) was also involved in the process. The addition of indomethicin to cultures improved the proliferative response in the presence of FDCs, indicating that inhibition was mediated, in part, by prostaglandins. These results indicate that FDCs regulate T-cell proliferation by two molecular mechanisms and that FDC:T-cell interactions may play a pivotal role in germinal center development.

Wide variation in serum anion gap measurements by chemistry analyzers.

The traditional anion gap [AG = Na-Cl-(total CO2)] mean value of 12 mEq/L was established during the 1970s with analyzer methods that are no longer used widely. No studies have systematically compared mean AG values from analyzers in current use. We used data from healthy subjects obtained from 27 clinical laboratories, 5 manufacturers, and 8 publications to compute mean AG values from 1970s analyzers and 8 current analyzers. We also compared mean AG values by evaluating Na, Cl, and total CO2 data from the College of American Pathologists Chemistry Surveys (1990-1996). Data from healthy subjects showed that overall mean AG values of the 9 analyzers ranged from 5.9 to 12.4 mEq/L. The pooled (i.e., average) AG SD was 2.3 mEq/L. We then used the data of the Surveys and the mean value from 1 analyzer to compute predicted mean values for the other 7 current analyzers. Almost all mean AG values predicted from the Surveys agreed (within 1.5 mEq/L) with mean values from healthy subjects. These results show that mean values of analyzers vary widely, indicating that analytic bias strongly influences the AG. The results should be a useful guide for the AG measurements that can be expected from different analyzers.

Oligoclonal protein bands and Ig isotype switching in multiple myeloma treated with high-dose therapy and hematopoietic cell transplantation.

Multiple myeloma (MM) is usually characterized by production of a single serum monoclonal protein of constant isotype and light-chain restriction. Multiple Ig isotypes and isotype switches, which are rare in untreated patients, are reported to be more common in patients undergoing myeloablative therapy. These additional protein bands, detected by immunofixation electrophoresis (IFE), could be due to altered paraprotein production by the malignant plasma cell clone or oligoclonal Ig production during recovery of B-cell function after myeloablative therapy. We analyzed abnormal protein bands (APB), distinct from the presenting paraprotein, in 550 patients receiving high-dose therapy with autologous hematopoietic cell transplantation at a single institution. Fifty-five patients (10%) had APB, 48 had oligoclonal bands (OB), and 23 had an apparent isotype switch (IS) on IFE (16 had both OB and IS). Morphologic and flow cytometric examination of bone marrow in 17 patients with IS showed no evidence of a clonal plasma cell isotype switch. Patients with APB had significantly higher complete response to therapy (67% v 37%, P = .001). To assess the independent prognostic relevance of APB, a multivariate analysis was performed among 471 patients surviving at least 12 months from first transplant (all patients developing APB had done so by 12 months from first transplant). APB (in 50 patients) was a favorable feature for both event-free (rank 3, P = .004) and overall survival (rank 3, P = .0005). We propose that OB and IS are likely to be due to recovery of Ig production rather than alterations in the biology of the malignant plasma cell clone.

Ionized magnesium concentrations in critically ill children.

OBJECTIVE: To test the hypothesis that many critically ill children exhibit ionized hypomagnesemia despite having normal total magnesium (TMg) concentrations. DESIGN: A prospective, observational study with convenience sampling. SETTING: Pediatric and cardiovascular intensive care units of a large children's hospital. PATIENTS: Patients aged 1 day to 21 yrs admitted from January 1 to October 31, 1996. Patients with chronic renal failure or weight <3 kg were excluded. A group of healthy children involved in a school-based nutritional assessment study were also studied. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Sixty-seven patients (5.4+/-5.7 [SD] yrs) and 24 healthy children (10.84+/-0.93 yrs, p< .001) were studied. Plasma was assayed for ionized magnesium (IMg) using a blood analyzer. Forty (59%)/67 critically ill subjects had IMg concentrations <0.40 mmol/L, the lowest published normal value and the lowest value observed in our group of healthy children. Of these, 24 (60%)/40 had normal TMg concentrations. IMg was significantly (p=.00) lower in critically ill subjects than in the group of healthy children (0.37+/-0.10 mmol/L vs. 0.46+/-0.03 mmol/L). IMg did not correlate strongly with ionized calcium (r2=0.49), albumin (r2=0.09), or pH (r2=0.18). CONCLUSION: Many critically ill children exhibit ionized hypomagnesemia with normal TMg concentrations. These children would not be recognized as magnesium-deficient based on routine TMg testing. Critically ill children exhibited significantly lower concentrations of IMg than a group of healthy children.

Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants.

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.

Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody.

Follicular dendritic cells (FDC) are found in germinal centers and may play an important role in germinal center (GC) B cell development. However, very little is known about FDC because it is very difficult to obtain a pure preparation of FDC. To aid in the purification of human FDC, we produced a mouse monoclonal antibody named HJ2 that immunohistochemically stains FDC in various lymphoid tissues. Isolated HJ2+ cells obtained by flow cytometric cell sorting exhibit morphological features associated with FDC and display numerous FDC-associated surface markers, but do not express T cell and B cell markers. Taken together, these findings indicate that HJ2 binds FDC and can be used to purify human FDC. Phenotypic studies of purified FDC revealed that FDC express large amounts of the complement (C)-regulatory proteins CD46, CD55, and CD59. By deactivating C, these proteins may safeguard FDC from membranolysis by surface-bound C containing immune complexes. To begin studies of FDC function, we cultured purified FDC and found they survive for at least 2 weeks in vitro. Furthermore, FDC were potent stimulators of allogeneic peripheral blood mononuclear cells and a T cell line in mixed lymphocyte cultures, and on a cell basis, FDC were more active than B cells. The purification of functionally active human FDC should facilitate studying the role of FDC in B cell development within GC.

Cytokine expression by germinal center cells.

Germinal centers (GC) primarily consist of B cells along with a small number of T cells (5 to 10%) and follicular dendritic cells (FDC) (< or = 1%). Although extensive Ag-driven B cell proliferation and maturation occurs in GC, very little is known about the role of cytokines in the development of GC B cells. Therefore, to identify cytokines present in the GC microenvironment that may influence B cell development, we systematically examined cytokine gene expression by GC cells. GC T cells (CD57+/CD4+), GC B cells (CD77+), and FDC (HJ2+) were isolated from human tonsils by cell sorting using a flow cytometer. Freshly isolated GC cells were examined for mRNA expression for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, and IFN-gamma using reverse transcription polymerase chain reaction. Freshly isolated GC T cells consistently expressed IL-4 mRNA (11 of 12 tonsils), whereas CD57- Th cells (mostly non-GC Th cells) were often negative for IL-4 mRNA. When the other nine cytokine mRNA were studied, freshly isolated CD57+ Th cells occasionally expressed mRNA for IL-10, TNF-alpha, and IFN-gamma. CD57- Th cells were occasionally positive for IL-1 beta, IL-10, IFN-gamma, and TNF-alpha, and negative for IL-2 and IL-6. Freshly isolated GC B cells as well as FDC failed to express detectable quantities of mRNA for all 10 cytokines that were studied. Thus, IL-4 is the only cytokine out of 10 that is consistently expressed in GC and may be important for the development of B cells in GC. After stimulation of CD57+ Th cells with PWM, production of IL-4 mRNA was dramatically reduced, whereas CD57- Th cell production of IL-4 was greatly augmented. This finding indicates that GC T cells may differ from other Th cells in cytokine gene expression and that results of cytokine production obtained after in vitro stimulation do not always reflect in vivo results.

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.

Germinal center T cells are distinct helper-inducer T cells.

Germinal centers (GCs) contain a significant number of CD4+ T cells, but what role these T cells may play in the development of GC B cells has not been determined. To gain insight into their role, we studied the phenotype of GC T cells and the lymphokines secreted by GC T cells isolated from human tonsils obtained after tonsillectomies. In addition to confirming that a large fraction of GC T cells are Leu-7(CD57)+ and Leu-8-, we found that they have no binding sites for peanut agglutinin. Furthermore, we found that they are CD45RA- and CD45R0+, the phenotype of helper-inducer T cells. We also found that Leu-7(CD57)+ cells display CD69, a phenotypic marker of very early cell activation, but do not display three other markers of cell activation: CD25 [interleukin-2 (IL-2) receptor], CD71 (transferrin receptor), and DR. When isolated, Leu-7(CD57)+ cells were stimulated in vitro with a mitogen that can induce peripheral blood T cells with the helper-inducer phenotype to produce various cytokines, Leu-7(CD57)+ cells did not produce IL-2, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) in significant amounts. Taken together, GC T cells from a distinct subpopulation of T cells with helper-inducer phenotype by their histologic location, by their surface phenotype, and by their ability to produce lymphokines. This finding is consistent with the possibility that GC T cells have been selectively recruited to actively help B cells develop in GCs.

Cytokine production by T helper cell subpopulations during prolonged in vitro stimulation.

In man, CD4+ T cells can be divided into phenotypically distinguishable subsets with different function whereas CD4+ T cells with the opposite pheno-CD45RO and low levels of CD45RA antigen provide help for mitogen-induced immunoglobulin production whereas CD4+ cells with the opposite phenotype suppress immunoglobulin production. However, studies examining cytokine production by phenotypically defined CD4+ T cell subsets have led to different conclusions. Further, very few studies have examined cytokine production by freshly isolated CD4+ T cell subsets during extended culture periods. Thus, we examined the production of several cytokines (at various time points) by CD4+ T cell subsets that were isolated in several ways, and stimulated with PWM, Con A, and PHA in a well-defined serum-free culture system. We found that CD4+, CD45RA- (or CD45RO+) T cells consistently produced the most IL-2, IFN-gamma, and TNF-alpha after mitogen stimulation for 2 days. PWM induced the largest quantities of each cytokine, although a similar pattern of production was observed in response to Con A and PHA. We were unable to detect IL-4 production by mononuclear cells and CD4+ T cell subsets suggesting that, if it is produced at all, IL-4 is produced in extremely small quantities. When the culture period of initially CD45RO- T cells was extended beyond 2 days, the culture supernatant contained increased quantities of each cytokine and the cells in the culture had an increased number of cells expressing CD45RO antigen. Together, these data indicate that CD4, CD45RA- (or CD45RO+) T cells in peripheral blood are the major producers of IL-2, IFN-gamma, and TNF-alpha following short-term mitogen stimulation, and that phenotypically defined peripheral blood T cell subsets do not maintain a distinct pattern of cytokines during extended culture periods.