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Background: Sexually transmitted infections including gonorrhea and chlamydia are common in the active-duty military population, with historically higher rates than their civilian counterparts. Prevention and screening are 2 of the main strategies used to reduce the chronic medical complications and costs associated with untreated gonorrhea and chlamydia; however, there is little information in the literature regarding treatment time after a positive screening. To our knowledge, there has not yet been a study regarding delayed treatment of gonorrhea and chlamydia in the active-duty population., Methods: We performed a population-based retrospective observational study on active-duty service members (ADSMs) diagnosed with gonorrhea and chlamydia from 2010-2019. Statistical analysis was performed to determine differences in treatment times for key demographics. This study was reviewed and approved by the Brooke Army Medical Center Institutional Review Board., Results: Average treatment time was 3.5 days for individuals with chlamydia and 5 days for those with gonorrhea. Treatment within 2 weeks was met for 94% of people diagnosed with chlamydia and 91% of people diagnosed with gonorrhea. Delay in treatment times for chlamydia were seen in men, ages 25-34, full-time active-duty service members, those with a history of prior infection, and soldiers in the Army. Gonorrhea treatment times were delayed in men, members of the Coast Guard, ages 35-44, and those with a history of prior infection., Conclusions: Significant differences in treatment time were seen based on sex, age, branch of service, rank, and history of prior infection., (Copyright © 2024 American Sexually Transmitted Diseases Association. All rights reserved.)

Purpose: The therapeutic effect of allogeneic hematopoietic stem cell transplantation (HSCT) for patients with myeloid malignancies has been attributed in part to a graft-versus-leukemia effect that is dependent on donor T lymphocytes. CD8(+) T-cell responses to MHC class I-restricted tumor epitopes, not just allogeneic antigens, may help mediate antileukemia effects after HSCT, but the specificity and function of such cells are not completely understood., Experimental Design: We examined the diversity, phenotype, and functional potential of leukemia-associated antigen-specific CD8(+) T cells in patients with myeloid leukemia following allogeneic HSCT. Screening for antigen-specific T cells was accomplished with a peptide/MHC tetramer library., Results: Patients with acute myelogenous leukemia or chronic myelogenous leukemia in remission following HSCT exhibited significant numbers of peripheral blood CD8(+) T cells that recognized varying combinations of epitopes derived from leukemia-associated antigens. However, these cells failed to proliferate, release cytokines, or degranulate in response to antigen-specific stimuli. As early as 2 months after HSCT, CD8(+) T cells from patients were predominantly CD28(-) CD57(+) and had relatively short telomeres, consistent with cellular senescence., Conclusions: Circulating leukemia-specific CD8(+) T cells are prominent in myeloid leukemia patients after HSCT, but such cells are largely functionally unresponsive, most likely due to replicative senescence. These findings carry important implications for the understanding of the graft-versus-leukemia effect and for the rational design of immunotherapeutic strategies for patients with myeloid leukemias.

Because natural killer (NK) cells can be potent anti-tumor effectors after allogeneic stem cell transplantation, we investigated NK reconstitution and receptor expression in patients undergoing allogeneic hematopoietic stem cell transplantation, focusing on the activating receptors that trigger anti-tumor responses. We determined that NK levels in the peri-transplant period were inversely proportional to the dramatic rise and fall in plasma levels of the NK homeostatic cytokine IL-15, which increased more than 50-fold from pretreatment to the day of transplant during the lymphoreductive preparative regimen. Furthermore, in NK cells cultured with IL-15, we observed an up-regulation of the activating receptors NKG2D, NKp30, and NKp46, associated with an increase in anti-tumor lytic activity. Similarly, the expression of these activating receptors increased significantly during the early post-transplant period, concurrent with a rapid increase in total NK cells and a shift toward increased expression of CD56. These data suggest that the cytokine milieu of transplants, in particular elevated levels of IL-15, may contribute to anti-tumor efficacy post-transplant by enhancing the recovery of NK subsets and modulating expression of activating receptors.

Paraneoplastic cerebellar degeneration (PCD) is linked to an immune response against cerebellar degeneration related antigen 2 (cdr2) co-expressed in tumor and Purkinje neurons. Here, comprehensive immune-assessment assays were used to analyze CD8(+) T cells from 7 PCD patients, but no evidence was found of CD8(+) T cells specific for either of two previously described cdr2 epitopes (cdr2-1 and cdr2-2). In contrast, viral-specific CD8(+) T cells from healthy volunteers and PCD patients were measurable. These findings are inconsistent with an obligate role for cdr2-1- or cdr2-2-specific CD8(+) T cells in the pathogenesis of PCD.

The human telomerase reverse transcriptase hTERT is overexpressed in most human tumors and contributes importantly to oncogenesis by maintaining the integrity of telomeric DNA. Despite being a self-antigen, the hTERT enzyme is immunogenic. Peptides derived from hTERT have been shown both in vitro and in vivo to drive the activation and proliferation of peptide-specific T lymphocytes. An HLA-A2-binding peptide from Htert (I540, ILAKFLHWL) has been used to generate peptide-specific T cells in vitro and in vivo in patients that lyse telomerase-positive tumors in an MHC-restricted fashion. Although these and other data suggest that I540 is naturally processed and presented on the surface of certain tumor cells, there are reports that I540-specific T cells, and in particular, T cell clones, do not lyse tumors in vitro. Here, we compared cytotoxic function of I540-specfic T cell clones vs. polyclonal T cell lines, including clones and lines generated from the same donor. We found that I540-specific polyclonal T cell lines lyse telomerase-positive tumors whereas non-specific polyclonal T cell lines and I540-specific T cell clones do not. Estimated TCR avidity for I540, as well as cell surface expression of CD45RO, CD45RA, CD28, CD27, CD57 and CD62L were similar between lines and clones. V beta usage, however, differed such that the majority of the I540-specific TCR repertoire found in polyclonal T cell lines was not represented in clones generated from the same source material. Thus, I540-specific T cells can vary in cytotoxic potential depending on the method of generation, isolation and expansion.

The human telomerase reverse transcriptase (hTERT) is nearly universally overexpressed in human cancer, contributes critically to oncogenesis, and is recognized by cytotoxic T cells that lyse tumors. CD8+ T cells specific for hTERT naturally occur in certain populations of cancer patients in remission, but it remains poorly understood whether such T cells could contribute to tumor immunosurveillance. To address this issue, we induced hTERT-specific T cells in vivo via peptide vaccination in 19 patients with metastatic breast cancer who otherwise had no measurable T-cell responses to hTERT at baseline. Tumor-infiltrating lymphocytes (TIL) were evident after, but not before vaccination, with 4% to 13% of postvaccine CD8+ TIL specific for the immunizing hTERT peptide. Induction of TIL manifested clinically with tumor site pain and pruritus and pathologically with alterations in the tumor microenvironment, featuring histiocytic accumulation and widespread tumor necrosis. hTERT-specific CD8+ T cells were also evident after vaccination in the peripheral blood of patients and exhibited effector functions in vitro including proliferation, IFN-gamma production, and tumor lysis. An exploratory landmark analysis revealed that median overall survival was significantly longer in those patients who achieved an immune response to hTERT peptide compared with patients who did not. Immune response to a control cytomegalovirus peptide in the vaccine did not correlate with survival. These results suggest that hTERT-specific T cells could contribute to the immunosurveillance of breast cancer and suggest novel opportunities for both therapeutic and prophylactic vaccine strategies for cancer.

Background & Aims: Acute hepatitis C virus (HCV) infection becomes chronic in the majority of patients. Although HCV-specific CD4 T-cell response is associated with HCV clearance, less is known about virus-specific CD8 T-cell or neutralizing antibody (nAb) responses and the role of CD4 help in their induction during acute infection., Methods: HCV-specific CD4, CD8, and HCV pseudoparticle (HCVpp) nAb responses were monitored in acutely HCV-infected patients to define their relative contributions to viral clearance., Results: Our results show that the outcome of acute hepatitis C is associated with a functional hierarchy in HCV-specific CD4 T-cell response and the scope of virus-specific, total T-cell interferon-gamma response. HCV-specific CD8 T-cell response was readily detectable in acutely HCV-infected patients regardless of virologic outcome or virus-specific CD4 T-cell response. In contrast, HCVpp-specific nAbs were readily detected in patients with chronic evolution and impaired virus-specific CD4 T-cell response but not in patients who cleared infection with robust virus-specific CD4 T-cell response., Conclusions: The outcome of acute hepatitis C is associated with efficient virus-specific CD4 T-cell response(s) without which HCV-specific CD8 T-cell and heterologous nAb responses may develop but fail to clear viremia. Furthermore, HCV-specific nAb responses may not be induced despite robust virus-specific CD4 T-cell response.

Purpose: Tumor immunosurveillance influences oncogenesis and tumor growth, but it remains controversial whether clinical failure of immunosurveillance is a result of lymphocyte dysfunction or tumor escape. In this study, our goal was to characterize the physiology of tumor immunosurveillance in children with high-risk neuroblastoma (HR-NBL)., Patients and Methods: Immunohistopathologic studies were carried out on 26 tumor samples from a cohort of HR-NBL patients diagnosed at Children's Hospital of Philadelphia for the 2-year period from May 2003 to May 2005. Blood from nine HLA-A2+ patients in this cohort was analyzed for T cells specific for the antiapoptotic protein survivin., Results: Survivin protein was expressed by 26 of 26 tumors. In HLA-A2+ patients, circulating cytotoxic T lymphocytes (CTLs) specific for survivin were detected by peptide/major histocompatibility complex tetramer analysis in the blood of eight of nine children with HR-NBL at the time of diagnosis. Rather than being selectively rendered anergic in vivo, circulating survivin-specific CTLs were highly functional as shown by cytotoxicity and interferon gamma enzyme-linked immunospot assays in six of nine patients. Survivin-specific CD107a mobilization by T cells was found in five of five patients. By immunohistochemistry, tumor-infiltrating T cells were few or absent in 26 of 26 tumors., Conclusion: Children with HR-NBL harbor robust cellular immune responses to the universal tumor antigen survivin at the time of diagnosis, but intratumoral T cells are strikingly rare, suggesting a failure of cellular immunosurveillance. Efforts to develop novel therapies that increase T-cell trafficking into tumor nests are warranted.

CD69 is thought to be a pluripotent signaling molecule expressed on the surface of a number of activated leukocytes including B, T, and NK cells, monocytes, neutrophils, and platelets. While some advances have been made regarding the mechanisms by which CD69 may participate in such diverse functions as cell aggregation, cellular cytotoxicity, and release of cytokines and inflammatory mediators, the most proximal links of signal initiation have not been identified. Our study has identified, by immunoprecipitation and direct protein sequencing (LC/MS/MS), binding of CD69 to an N-terminal protein fragment of calreticulin expressed on the cell surface of human PBMCs. Given the recently identified roles calreticulin plays in cell adhesion and angiogensis, the identification of CD69 binding directly to calreticulin may provide insights into mechanism(s) by which CD69 or other CD69 family members, i.e., LLT1 and AICL participates in such diverse functions.

Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4(+) T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA(+)CD62L(+) and signal-joint TCR rearrangement excision circle-bearing CD4(+) populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4(+) T cell populations. This recovery encompassed the recovery of normal CD4(+) T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vbeta diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.

Breast cancer cells pose a difficult target for detection due to their lack of tumor specific markers. Small numbers of metastatic cells circulate through the blood and reach target organs, but current methods are presently not sensitive enough to allow for early detection. Our goal is to develop a method for specific and sensitive detection for breast cancer tumor cells by using cell enrichment coupled to RT-PCR with selective primer pairs. Fifty million normal donor mononuclear cells (NDMNC) were spiked with serial dilutions of a breast cancer cell line, positively selected for surface expression of the epithelial cell adhesion molecule (EPCAM) with anti-Ber-EP4 mAb. Total RNA was isolated and the cDNA was transcribed and measured by quantitative PCR for cytokeratin 19 (CK19), epidermal growth factor receptor (EGF-R), and beta-2-microglobulin (beta2M) transcripts using specific hybridization probes with the LightCycler System. A sensitivity of 1 tumor cell in 5 million NDMNC was consistently achieved with a metastatic breast cancer cell line with target primer sets for CK19 and EGF-R. Less, but appreciable sensitivity was achieved by spiking NDMNC with other breast cancer cells. Detection of both gene targets ranged between 1 in 5,000 to 5 million cells. The method described, a cell enrichment procedure and RT-PCR using specific hybridization probes (spanning introns and hybridizing to areas that discriminate transcribed pseudogenes), results in increased sensitivity while decreasing false positives above those methods previously reported. This general approach may have wide applicability to increasing sensitivity of minimal residual disease detection in other cancers.

Vaccination with antigen-presenting cells (APCs) engineered to mimic mechanisms of immune stimulation represents a promising approach for cancer immunotherapy. Dendritic cell vaccines have entered phase 3 testing in adult malignancies, but such vaccines in children have been limited. We demonstrate that CD40-activated B cells (CD40-B) transfected with RNA may serve as an alternative vaccine that can be generated from small blood volumes regardless of patient age. CD40-B from pediatric patients are efficient APCs and can be loaded with RNA as an antigenic payload, permitting simultaneous targeting of multiple antigenic epitopes without the necessity of HLA matching. For viral and tumor antigens, CD40-B/RNA technology induced cytotoxic T lymphocytes (CTLs) from adults and children, which could be identified with peptide/major histocompatibility complex (MHC) tetramers. These CTLs secreted interferon-gamma (IFN-gamma) and killed targets in an MHC-restricted fashion. For pooled neuroblastoma RNA and autologous neuroblastoma RNA, CTLs that lysed neuroblastoma cell lines, including CTLs specific against the widely expressed tumor-antigen survivin, were generated. These findings support a novel platform for tumor-specific vaccine or adoptive immunotherapies in pediatric malignancies.

This report investigated in vivo turnover kinetics of marrow hematopoietic progenitors and precursors using a recently developed stable isotope-mass spectrometric technique (SIMST). Human subjects were administered a 2-day infusion of 6,6-[2H2]-glucose, a nontoxic stable isotope-labeled form of glucose, which becomes incorporated into DNA of all S-phase cells. The percent [2H2]-glucose incorporated into DNA in the form of [2H2]-deoxyadenosine (%[2H2]-dA enrichment) was determined by gas chromatography-mass spectrometry. The rate constant of replacement of unlabeled by labeled DNA strands (labeling kinetics) was used to calculate population turnover kinetics of CD34+ cells, CD133+ cells, and CD133-CD34+ cells. The observed mean replacement half-life (t1/2) was 2.6 days for CD34+ cells, 2.5 days for CD133-CD34+ cells, and 6.2 days for CD133+ cells. Results from the estimated rate constant of replacement of labeled by unlabeled DNA (delabeling kinetics) also demonstrated slower turnover rates for CD133+ cells than for CD133-CD34+ cells. Although there was a relatively rapid initial decrease in the %[2H2]-dA enrichment, low levels of labeled DNA persisted in CD34+ cells for at least 4 weeks. The results indicate the presence of subpopulations of CD34+ cells with relatively rapid turnover rates and subpopulations with a slower t1/2 of 28 days. Results also demonstrate that in vivo [2H2]-glucose-SIMST is sensitive enough to detect differences in turnover kinetics between erythroid and megakaryocyte lineage cells. These studies are the first to demonstrate the use of in vivo [2H2]-glucose-SIMST to measure in vivo turnover kinetics of subpopulations of CD34+ cells and precursors in healthy human subjects.

The early activation marker, CD69, is transiently expressed on activated mature T cells and on thymocytes that are undergoing positive or negative selection in the thymus. CD69 is a member of the NK gene complex family of C-type lectin-like signaling receptors; however, its function is unknown. In this report, we describe the characterization of mice that constitutively express high levels of surface CD69 on immature and mature T cells throughout development. Constitutive surface expression of CD69 did not affect T cell maturation, signaling through the TCR or thymocyte selection. However, phenotypically and functionally mature thymocytes accumulated in the medulla of CD69 transgenic mice and failed to be exported from the thymus. The retention of mature thymocytes correlated with transgene dose and CD69 surface levels. These results identify a potential role for CD69 in controlling thymocyte export, and suggest that the transient expression of CD69 on thymocytes and T cells may function to regulate thymocyte and T cell trafficking.

Human CD69 is uniquely glycosylated at typical (Asn-X-Ser/Thr) and atypical (Asn-X-Cys) motifs, which represents the molecular basis for the formation of CD69 homodimers and heterodimers. Here we examined the importance of N-glycosylation for the assembly and intracellular transport of CD69 proteins using mutant CD69 molecules that specifically lack typical and atypical N-glycan attachment motifs. These studies verify the importance of Cys residues in atypical triplet sequences for N-glycan addition to human CD69 proteins in the endoplasmic reticulum (ER). In addition, these data demonstrate that monoglycosylated CD69 proteins (bearing N-glycans exclusively at atypical or typical sites) and aglycosylated CD69 molecules (lacking N-glycans) efficiently dimerize in the ER and have similar stability as wild-type CD69 molecules. Finally, these results show that CD69 proteins lacking atypical or typical N-glycan addition sites are transported to the plasma membrane., (Copyright 1999 Academic Press.)

CD69 is expressed on the surface of all hematopoietically derived leukocytes and is suggested to function as a multipurpose cell-surface trigger molecule important in the development and activation of many different cell types. Human CD69 contains only a single consensus sequence for N-linked oligosaccharide addition within its extracellular domain (Asn-Val-Thr), yet exists as two distinct glycoforms that are assembled together into disulfide-linked homodimers and heterodimers. The molecular basis for human CD69 heterogeneity has remained elusive. In the current report we show that human CD69 glycoforms are generated before the egress of CD69 proteins from the endoplasmic reticulum to the Golgi and are synthesized under conditions where Golgi processing is inhibited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation site in the Golgi complex. Importantly, these data demonstrate that contrary to current belief, not one but two sites for N-glycan addition exist within the human CD69 extracellular domain and identify the second, "cryptic" CD69 N-glycan attachment site as the atypical Cys-containing glycosylation motif, Asn-Ala-Cys. The results in this study provide a molecular basis for human CD69 heterogeneity and show that multiple dimeric forms of human CD69 result from the variable addition of N-glycans to atypical and typical glycosylation motifs within the CD69 extracellular domain.

Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.

Human PBL express one or more of the three classes of Fc receptors for IgG (Fc gamma receptors, I, II, and III). Each type of Fc gamma receptor has a characteristic binding pattern for isotypes of human and mouse IgG. Large granular lymphocyte/NK cells (LGL/NK cells) express the transmembrane form of Fc gamma RIII (Fc gamma RIIIa) on their surface, whereas polymorphonuclear neutrophils (PMN) express the glycosyl phosphatidyl inositol-linked receptor that is the product of the Fc gamma RIIIB gene. Fc gamma RIII has been reported to have low affinity for monomeric IgG, regardless of the cell type on which it is expressed. This study demonstrates specific and saturable binding of monomeric human IgG (M-IgG) to Fc gamma RIIIa on LGL/NK cells but not to Fc gamma RIIIb on PMN. M-IgG binding revealed an expression polymorphism between individuals, those with either high (12,000 to 15,000) or low (4,000 to 7,000) binding sites per cell. Both high and low M-IgG-binding LGL/NK cells bound hulgG1 and IgG3 but did not bind IgG2 or IgG4. The high/low Fc gamma RIIIA expression polymorphism correlated with the ability of an individual's LGL/NK cells to perform antibody-dependent cellular cytotoxicity of erythroid cell targets.

The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential (Vbl = -69 mV), a higher relative potassium conductance (tK = 0.6), lower intracellular Na+ activity (ANa = 18.4 mM), and higher intracellular K+ activity (AK = 67.8 mM). No evidence for a conductive sodium-dependent or sodium-independent HCO3- pathway could be demonstrated. The basolateral Na-K pump is inhibited by 10(-4) M ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results in ANa = 64.8 mM, AK = 18.5 mM, and Vbl = -28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K+ conductance, as evaluated by increasing bath K+ to 17 mM, is dependent upon the resting Vbl in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na+ and K+ gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment.

The human monocyte and macrophage Fc receptor that binds human IgG with high affinity is a surface glycoprotein with a relative molecular mass of approximately 70 kDa. This receptor (Fc gamma RI) has been partially characterized using mAb 32 which binds outside the Fc binding domain of the receptor, but nonetheless triggers Fc receptor-dependent functions. In this study, we describe the properties of four new antibodies with specificity for Fc gamma RI. Based on additivity and cross-blocking studies, we conclude that two of these antibodies (mAb 22 and 44) define a third epitope which is distinct from the binding sites for both mAb 32 and the Fc portion of human IgG. Each Fc gamma RI-specific hybridoma was selected for stable sublines expressing high levels of mAb on the cell surface, and then tested for the ability of this surface mAb to trigger antibody-dependent cell cytotoxicity. All sublines were killed by human monocytes when used as targets in a 51Cr-release assay, whereas hybridomas specific for myeloid Ag other than Fc gamma RI were not killed. We conclude that Fc receptor function is triggered through binding to each of the three epitopes of Fc gamma RI that we have defined. These mAb will be useful for additional characterization of Fc gamma RI, and may, when incorporated into tumor-directed heteroantibodies, enhance tumor cell killing by human monocytes and macrophages.

Two metalloproteinases, collagenase and stromelysin, are produced in large quantities by synovial fibroblasts in individuals with rheumatoid arthritis. These enzymes play a major role in the extensive destruction of connective tissue seen in this disease. In this study, we show that heat shock of monolayer cultures of rabbit synovial fibroblasts increases expression of mRNA for heat shock protein 70 (HSP-70), and for collagenase and stromelysin. We found that after heat shock for 1 h at 45 degrees C, the mRNA expression for HSP-70 peaks at 1 h and returns to control levels by 3 h. Collagenase and stromelysin mRNA expression is coordinate, reaching peak levels at 3 h and returning to control levels by 10 h. The increase in mRNA is paralleled by an increase in the corresponding protein in the culture medium. 3 h of heat shock at a lower temperature (42 degrees C) is also effective in inducing collagenase and stromelysin mRNAs. Concomitant treatment with phorbol myristate acetate (PMA; 10(-8) or 10(-9) M) and heat shock is not additive or synergistic. In addition, all-trans-retinoic acid, added just before heat shock, prevents the increase in mRNAs for collagenase and stromelysin. Our data suggest that heat shock may be an additional mechanism whereby collagenase and stromelysin are increased during rheumatoid arthritis and perhaps in other chronic inflammatory stress conditions.

Interferon-gamma (IFN-gamma) activation of human monocytes in vitro results in enhanced phagocytosis and cellular cytotoxicity. These enhanced effector functions are attributable, at least in part, to increased expression of recognition molecules on the plasma membrane. In this article we report a rapid screening procedure for the primary selection of monoclonal antibodies (mAbs) which bind to cell surface molecules, the expression of which is increased or decreased by IFN-gamma. The procedure is based on flow cytometric analysis of mixed cell populations. Mouse mAbs were prepared using human monocytes cultured for 40 h with 400 U/ml of IFN-gamma as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-gamma for 40 h. Cells included in the mixture were chosen for their distinctive light scatter profiles. mAbs of interest were identified by preferential binding to monocytes and increased or decreased binding to monocytes treated with IFN-gamma. This procedure allowed us to screen several hundred clones per day, and to immediately eliminate mAbs that bound to B cells, T cells, neutrophils, and several cell lines. We selected ten mAbs which bound to surface molecules on monocytes that were modulated by IFN-gamma. Further characterization of five of the initial ten mAbs revealed that mAb gamma M phi 22.2 and mAb gamma M phi 197.1 bind to the high affinity Fc receptor for IgG (Fc gamma RI). mAb gamma M phi 28.3 appears to bind to a class II histocompatibility antigen and mAb gamma M phi 150.3 and mAb gamma M phi 195.18 appear to have binding patterns to human leukocytes and cell lines which are distinct from previously described mAbs. This rapid and specific procedure for screening mAbs has broad application for selecting mAbs that are specific for any given cell type and/or for surface molecules that are modulated by any cytokine and other hormone.