Allogeneic Serum Research Articles

In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability

Human bone marrow mesenchymal stem cells (hMSCs) represent an appealing source of adult stem cells for cell therapy and tissue engineering, as they are easily obtained and expanded while maintaining their multilineage differentiation potential. All current protocols for in vitro culture of hMSCs include fetal bovine serum (FBS) as nutritional supplement. FBS is an undesirable additive to cells that are expanded for therapeutic purposes in humans because the use of FBS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. In the present study, we have therefore investigated if autologous serum (AS) or allogeneic human serum (alloHS) could replace FBS for the expansion of hMSCs in vitro. We discovered that the choice of serum affected hMSCs at several different levels. First, hMSCs in AS proliferated markedly faster than hMSCs in FBS, whereas use of alloHS resulted in hMSC growth arrest and death. Second, hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in AS. Interestingly, genome-wide microarray analysis identified several transcripts involved in cell cycle and differentiation that were differentially regulated between hMSCs in FBS and AS. Finally, several transcripts, including some involved in cell cycle inhibition, were upregulated in hMSCs in FBS at a late passage, whereas the hMSC transcriptome in AS was remarkably stable. Thus, hMSCs may be expanded rapidly and with stable gene expression in AS in the absence of growth factors, whereas FBS induces a more differentiated and less stable transcriptional profile.

Human Serum for Culture of Articular Chondrocytes

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without losing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.

Serological identification of breast cancer-related antigens from aSaccharomyces cerevisiae surface display library

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies.

IgE-Dependent Activation of T cells by Allergen in Atopic Dermatitis: Pathophysiologic Relevance

The importance of interactions between allergen and IgE in allergen-mediated activation of T lymphocytes from patients with atopic dermatitis (AD) is unclear. A role for this interaction is implied by past evidence for IgE-facilitated presentation of allergen to T cells, but this phenomenon has only been demonstrated in specific in vitro systems biased to maximize the effect. It is therefore not known whether the process is relevant in patients. We now show that the responses to allergen of unmodified peripheral blood mononuclear cells (PBMC) from individual AD patients are significantly greater in the presence of fresh, unheated, IgE-containing autologous serum than the same serum heated under IgE-denaturing conditions or specifically depleted of IgE by immunoprecipitation. In six independent experiments, 59%-67% of the maximal in vitro PBMC response to allergen was found to be dependent upon the presence of IgE in autologous serum used at 5% final concentration. These data provide the first evidence that sufficient amounts of allergen-specific IgE and allergen-reactive T cells occur concomitantly in the blood of individual AD patients to allow IgE-enhanced T cell responses to allergen. We conclude that IgE-enhanced T cell responses are pathophysiologically relevant and a therapeutic target in AD.

Retroviral Gene Transfer of Suicide Genes in Human T Lymphocytes: Improved Transduction Conditions for a Clinical Protocol.

Aiming to control GVHD by means of the transfer of suicide genes into donor T cells, we seeked to optimise the transduction of human T cells using immobilized anti-CD3i/28i MoAbs for T cell stimulation. In previous studies we showed that reducing the concentration of anti-CD3i, from 1,000ng/ml down to 1ng/ml, helps to preserve the cytotoxicity of the samples to an allogeneic stimulus and maintains the susceptibility of the T cells to the retroviral transduction. Aiming to reduce the cost of the manipulation procedure, we also tried to reduce the amount of anti-CD28 MoAb during the activation (pre-infection) and expansion (post-infection) steps. To investigate this issue, different doses of anti-CD28i were combined either with 1,000 ng/ml or with 10 ng/ml of anti-CD3i. In contrast to the effects observed with anti-CD3i, lowering the dose of anti-CD28i, from 1,000 ng/ml to a range of 500 to 10 ng/ml significantly decreased the transduction efficacy, as well as the total number of transduced T cells, regardless of the concentration of anti-CD3i used in the experiments. With the purpose of minimizing potential effects mediated by retroviral supernatants on the viability and growth of the T cells, we investigated whether standard infections with supernatants could be replaced by alternative or complementary transduction procedures based on the pre-loading of the retroviral vectors in fibronectin-coated bags. In these experiments, fibronectin-coated cell culture bags were pre-loaded with retroviral vectors by means of a 3h-incubation with the infective supernatants. Thereafter, peripheral blood lymphocytes were added to the culture bags, incubated O/N and then subjected to further infection cycles, either with pre-loaded vectors or with infective supernatants. Performing only one cycle of infection with either method, rendered similar low yields of transduced cells compared to results obtained after 2 infection cycles. Under these conditions, we found no differences between both methods, neither with respect to the transduction efficacy nor with the total number of transduced cells. Conducting a third infection cycle, either using the pre-loading or the supernatant infection method, did not increase significantly the transduction efficacy of the samples. Also, to simplify and improve the safety of the manipulation procedure, we studied alternatives to the use of human allogeneic or autologous serum for the supplement of the cell culture medium (RPMI). In these experiments, RPMI supplemented with 10% autologous serum was replaced by the CellGro and X-Vivo 10 serum-free media. Similar transduction efficacies and similar number of transduced cells were generated as a result of the manipulation of the samples with either medium. We conclude that optimised conditions of T cell stimulation and gene expression are obtained with 1ng/ml anti-CD3i and 1,000 ng/ml anti CD28i, and that conventional supernatant infections conducted with serum-supplemented media can be partially or completely replaced by pre-loading infections in serum-free media. We propose these manipulations to simplify and to reduce risks and costs associated to the transduction of human T cells; something that is of particular interest for the development of clinical protocols that require the infusion of large numbers of transduced T lymphocytes.

PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM), a malignant astrocytic tumour, represents the most frequent tumour of the human brain. Nevertheless, its molecular pathology is not well understood. We utilized the immune system, which contributes to cancer protection, to help identify new GBM-related genes. By screening a human GBM cDNA library with autologous patient serum (SEREX-approach), we isolated a gene termed PHF3 (PHD finger protein 3). The gene product of PHF3 is immunogenic in GBM as tested in an allogenic patient serum screening demonstrating antibodies in 24 of 39 (61.53%) sera, whereas none of the 14 healthy persons had antibodies against PHF3. While previous SEREX studies revealed allogenic antibody responses up to 40%, our results for PHF3 represent the highest reported rate for a specific antibody response. We show that GBM patients with an antibody response against PHF3 show significant better survival than patients without PHF3-specific antibodies. Because the amino acid sequence of PHF3 contains a PHD finger (also termed LAP motif), a TFIIS homology, a proline rich region and nuclear localization signals, it supposedly functions as a transcription factor. A polyclonal antibody generated against PHF3 shows nuclear expression in most investigated formalin-fixed, paraffin embedded tissues. In GBM, PHF3 expression is concentrated in cells surrounding necroses.

Gene Cloning of Immunogenic Antigens Overexpressed in Pancreatic Cancer

The serological analysis of recombinant cDNA expression libraries (SEREX) by utilizing a library derived from a human pancreatic adenocarcinoma cell line and IgG antibodies from an allogeneic patient serum led to the identification of 18 genes: 13 of these were known genes, and 5 were unknown genes. In Northern and RT-PCR analyses, we found that the expression of mRNA of 14 genes was elevated in pancreatic cancer cell lines compared with the levels in normal pancreatic tissues. In addition, the expression of mRNA of hsp105 in colon cancer was greater than that in normal colon tissue. Immunohistochemical analysis using anti-hsp105 antibody revealed that an increased expression of hsp105 is a characteristic feature of pancreatic ductal and colon adenocarcinoma. Furthermore, hsp105 immunoreactivity in some cases of gastric, esophageal, and hepatocellular carcinoma was much stronger than that in normal corresponding tissues. These molecules identified may provide good diagnostic markers for cancer cells.

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy

There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials. PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number. In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Reversal of the vasoconstrictor step of hyperacute xenogeneic rejection of the liver by endothelin antagonists.

The role of endothelin in the initial vasoconstrictor step of hyperacute xenogeneic rejection was investigated. Isolated rat livers were perfused in recirculation. Perfusion with human sera provided an ex vivo model of hyperacute rejection in a discordant combination. Perfusion of 10% xenogeneic serum induced a marked (70%) and sustained reduction of the liver flow and induced the release of endothelin into the perfusion medium. In contrast, perfusion of 10% allogeneic serum or of 10% decomplemented human serum induced a weak (25%) and transient reduction of the liver flow and induced the release of minimal amounts of endothelin. The simultaneous administration of BQ 123 and BQ 788, the respective antagonists of ET(A) and ET(B) endothelin receptors, or that of bosentan, a mixed ET(A)/ET(B) antagonist, antagonized the vasoconstrictor effect of 10% xenogeneic human serum, as well as that of 10(-9) M endothelin-1. The vasoconstrictor effects of xenogeneic serum on liver circulation are, at least partly, mediated through the release of endothelin by the graft.

Alternatives to animal sera for human bone marrow cell expansion: human serum and serum-free media.

The increasing use of cultured human cells in clinical trials is highlighting the need for alternatives to media containing animal sera that are typically used to support these cultures. Perfused cultures of BM mononuclear cells (MNC) were used to evaluate animal sera alternatives with respect to the output of primitive, progenitor, and stromal cells. A serum level of 20% was optimal, and this could be provided by FBS alone or by a mixture of horse serum (HoS) and FBS, but not by HoS alone. Allogeneic human plasma (20%) supported half the level of cell, CFU-GM, and LTC-IC output as compared with animal sera-containing control. Significant donor-to-donor variability in human plasma was observed, but this was mitigated by pooling of plasma samples. Autologous and allogeneic human plasma performed equivalently. The use of autologous or allogeneic human serum was found to be equivalent to the use of human plasma, but all were inferior to animal sera. Animal sera supported typical stroma and cobblestone formation, whereas stroma in human serum cultures was less dense. Eight commercial serum-free media were tested and found to support MNC expansion to varying degrees, but none approached the performance of the animal serum-containing control, particularly with respect to stromal (i.e., CFU-F) support. In fact, when MNC were cultured in parallel with CD34-enriched cells, output (from MNC) was higher only in control medium, apparently because serum-free media reduced accessory cell effects. Because of these results, a new serum-free medium was developed for MNC cultures. This formulation outperformed all commercial serum-free media, resulting in cell and LTC-IC output equivalent to that of control. However, CFU-GM and CFU-F output were 66% and 9% of control, respectively. Precoating the culture surface with collagen increased CFU-F (and Thy-1+ cell) output to control levels, although CFU-GM output was still lower than control. The addition of either fibronectin or PDGF had no measurable effect, nor did the use of 5-100-fold greater concentrations of growth factor supplementation. The serum-free medium also increased CD41+ and CD61+ cell output to 150%-220% of control levels. The development of this new serum-free medium has potential for use in the perfused BM MNC culture systems currently in clinical trials to test the efficacy of expanded cells after cytoablative chemotherapy.

Identification of multiple cancer/testis antigens by allogeneic antibody screening of a melanoma cell line library.

Cancer/testis (CT) antigens-immunogenic protein antigens that are expressed in testis and a proportion of diverse human cancer types-are promising targets for cancer vaccines. To identify new CT antigens, we constructed an expression cDNA library from a melanoma cell line that expresses a wide range of CT antigens and screened the library with an allogeneic melanoma patient serum known to contain antibodies against two CT antigens, MAGE-1 and NY-ESO-1. cDNA clones isolated from this library identified four CT antigen genes: MAGE-4a, NY-ESO-1, LAGE-1, and CT7. Of these four, only MAGE-4a and NY-ESO-1 proteins had been shown to be immunogenic. LAGE-1 is a member of the NY-ESO-1 gene family, and CT7 is a newly defined gene with partial sequence homology to the MAGE family at its carboxyl terminus. The predicted CT7 protein, however, contains a distinct repetitive sequence at the 5' end and is much larger than MAGE proteins. Our findings document the immunogenicity of LAGE-1 and CT7 and emphasize the power of serological analysis of cDNA expression libraries in identifying new human tumor antigens.

SSX: a multigene family with several members transcribed in normal testis and human cancer.

Analysis of t(X;18) translocation in synovial sarcoma had previously led to the definition of the SSX2 gene, the fusion partner on chromosome X. Subsequent screening of testicular cDNA libraries identified 2 highly homologous genes, SSX1 and SSX3. Among these 3 genes, SSX2 has been found to be identical to HOM-MEL-40, which codes for an immunogenic tumor antigen expressed in various human cancers. SSX2 thus belongs to the family of cancer/testis (CT) antigens, i.e., immunogenic protein antigens with characteristic mRNA expression in normal testis and in cancer. To define additional CT antigens, we have immuno-screened a testicular cDNA expression library with an allogeneic serum from a melanoma patient, and both SSX2 and SSX3 were isolated. Further studies using testicular cDNA and SSX probes defined 2 new members of this gene family, SSX4 and SSX5, while a shorter cDNA variant of SSX4 was also identified. All 5 members of the SSX family shared strong sequence homology, with nucleotide homology ranging from 88 to 95% and amino acid homology ranging from 77 to 91%. Genomic cloning of a prototype SSX gene (SSX2) showed that its coding region is encoded by 6 exons, and the shortened form of SSX4 cDNA represents an alternatively spliced product lacking the 5th exon. Analysis of SSX mRNA expression by gene-specific RT-PCR confirmed that all 5 SSX genes are expressed in testis. In addition, analysis of a panel of 12 melanoma cell lines showed strong mRNA expression of either SSX1 (3/12), SSX2 (3/12), SSX4 (1/12), or SSX5 (1/12), indicating variable activation of the genes in malignant cells.

Erratum: Benefits of blood cell transplant cryopreservation with oxypolygelatine (Gelifundol) plasma substitute

In two groups of 11 patients with poor prognosis malignancies undergoing high-dose sequential chemotherapy, we have evaluated the cryopreservation of blood cell transplants with oxypolygelatine-containing (55% oxypolygelatine, 6% hydroxyethylstarch, 5% dimethyl sulfoxide) vs standard human serum-containing (55% human serum, 6% hydroxyethylstarch, 5% dimethyl sulfoxide) cryoprotectant mixtures. Evidence is presented demonstrating that substitution of human serum proteins with oxypolygelatine has no detrimental effect either in vitro on the post-thawing recovery of hematopoietic progenitors or in vivo on the capacity of marrow reconstituting function in patients treated with myeloablative cancer therapy and autologous blood cell transplant. Oxypolygelatine is commercially available for clinical use as a plasma expander, is 30-fold less expensive than human serum albumin, is certified free of foreign serum proteins and antibodies as well as free of pyrogen, viral, mycoplasmal and bovine spongiform encephalopathy contaminants. Because of these characteristics, oxypolygelatine permits avoidance of: (1) the use of expensive serum albumin; (2) the fastidious preparation of autologous plasma or serum, and (3) the risk of infection associated with the infusion of allogeneic serum. Because of these practical advantages, we recommend the clinical use of oxypolygelatine as a substitute for human serum proteins for the routine cryopreservation of blood cell transplants.

Generation of in vitro natural cytotoxicity of horse lymphocytes against sarcoid-derived tumor cells not expressing major histocompatibility complex antigens

Abstract Objective To analyze in vitro lymphocyte-mediated immune responses of horses with sarcoids against allogeneic sarcoid cells containing endogenous retrovirus but not expressing major histocompatibility complex antigens. Design Lymphocyte-mediated immune reactions were assessed by means of proliferative responses in mixed lymphocyte tumor cell culture (MLTC) assay and lymphocyte-mediated cytotoxicity against various equine target cells. Animals 12 horses with sarcoid tumors and 15 control horses. Procedure Blood lymphocytes were cocultured in MLTC with allogeneic sarcoid cells (Mc-1, BayMc-1), equine testis cells, or normal equine dermal fibroblasts. Lymphocytes were assayed for proliferative responses by [3H]thymidine uptake and for cytotoxicity against the same targets by 51Cr release assay. The lymphocyte populations were analyzed for some common surface markers. Results Lymphocytes from horses with sarcoids exerted an anamnestic proliferative response in MLTC against Mc-1 cells, but this procedure never generated cytotoxic lymphocytes. However, lymphocytes from all horses cultured in medium with 10% allogeneic serum only had selective, natural cytotoxicity against Mc-1 that was generated without DNA synthesis. Approximately 80% of the lymphocytes disappeared during culture; however the remaining population of small, viable lymphocytes indicated a decrease of CD4+ and CD8+ T lymphocytes, but numbers of T cells with receptors for Helix pomatia A hemagglutinin were unaffected. Few lymphocytes had Fc-receptors for IgG, were complement-reactive positive cells, or were B cells expressing surface immunoglobulin. Conclusions Results may indicate a natural defense system, which preferentially recognizes and lyses tumor cells that are deficient in surface expression of major histocompatibility complex antigens, without intervention of conventional T-cell receptors or antibodies. (Am J Vet Res 1996;57:992–999)

Halogenated aromatic hydrocarbon-induced suppression of the plaque-forming cell response in B6C3F1 splenocytes cultured with allogenic mouse serum: Ah receptor structure activity relationships

The immunsuppressive effects of halogenated aromatic hydrocarbons (HAHs) were investigated in B6C3F1 female mice and in mouse splenocytes cultured with allogenic mouse serum using the Mishell-Dutton model for in vitro immunization to trinitrophenyl-lipopolysaccharide (TNP-LPS). Exposure to 2,3,7,8-tetrachlorodibenzo- P-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), 1,2,3,7,8-PeCDF, 1,3,6,8-tetrachlorodibenzofuran (TCDF), 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB), or 3,3′,4,4′,5,5′-hexaCB resulted in a dose-dependent suppression of the splenic plaque-forming cell (PFC) response both in vivo and in vitro. The effective dose required to decrease 50% (ED 50) of the response to 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF, 1,3,6,8-TCDF, 3,3′,4,4′,5-pentaCB, or 3,3′,4,4′,5,5′-hexaCB in vivo was 14.1, 5.5, 1695, 34800, 21, and 19 nmol/kg, respectively, and in vitro was 7.0, 10.6, 149, 2325, 9.1 and 9.1 nM, respectively. There was an excellent rank order and linear correlation between the in vivo versus in vitro activities for these HAHs ( r < 0.99) and the relative immunosuppressive potencies of these compounds paralleled their binding affinities for the aryl hydrocarbon (Ah) receptor. These results show that splenocytes cultured with allogenic mouse serum is an Ah-responsive in vitro assay which can be used for quantitating the immunosup-pressive effects of HAHS.

Identification of tumor-associated antigens in human hepatocellular carcinoma by autoantibodies

Hepatocellular carcinoma (HCC) is a major cause of death in Asian countries. The false-negative rate with serum alpha-fetaprotein level alone can reach 40% for early stage HCC patients. Due to the lack of sensitive and specific tumor markers for early diagnosis, it is impossible for HCC patients to receive effective therapy. However, tumor antigens can be recognized by immune cells and be rejected in immune responses. In order to identify antigens which may be used as new markers and immunotherapy targets for HCC, a cDNA expression library derived from an HCC sample was constructed, which was screened with mixed autologous and allogenic serum of HCC patients. Seventeen different HCC antigens were obtained, which are classified as tumor-associated antigens. A panel of allogenic sera from patients with chronic hepatitis, liver cirrhosis, HCC and other tumor entities and sera from health volunteers, was used for frequency analysis of antibody responses. Four of 17 antigens, including eukaryotic translation initiation factor 3, subunit I, lactate dehydrogenase 1, A chain, replication factor C2, 40 kDa and mitochondrial carrier triple repeat 1, reacted predominantly with sera from patients with HCC (31.8, 45.5, 27.3 and 50.0% respectively). Patients (81.8%) with HCC had the antibody against at least one of these four antigens, which indicates that disease-specific humoral response against these antigens was induced in HCC patients and the corresponding antibodies may be used as tumor markers for HCC.

On the possible mechanisms and predictability of clozapine-induced agranulocytosis.

Studies were conducted on serum removed from 15 patients before, during, and after, clozapine-induced agranulocytosis. Cytotoxic studies were compared with samples taken from patients during treatment with clozapine who did not develop agranulocytosis or treatment controls (TC); additional controls consisted of allogeneic (NC) and autogeneic serum from apparently normal people. The effect of serum on measurable functions of polymorphonuclear neutrophils (PMNs) taken from normal people was tested. Procedures under study included suppression of post-phagocytosis-induced 14CO2-indicated respiratory burst, as well as ejection of trypan blue by test PMNs. PMNs exposed to active agranulocytosis serum plus complement displayed diminished 14CO2 emission during phagocytosis or failed to eject trypan blue. PMNs exposed to serum of TC and NC continued to function normally as regards 14CO2 emission and trypan blue ejection. Five patients studied before the development of agranulocytosis showed suppressed PMN function, which increased to peak value during agranulocytosis and then disappeared within 40 days of recovery. Similar suppression of colony forming units of granulocytes and macrophages (CFU-GM) was found whenever agranulocytosis serum was included in the marrow culture. The cytotoxic material required complement for its full expression, was not dialysable, was neutralised by anti-IgM serum, and was absorbed by test PMNs. Furthermore, solutions of clozapine or 5 of its metabolites offered no similar suppression of PMN function in vitro after incubation in an aqueous medium or with normal serum. These observations favour development of an immunogenic clone in sensitive people during active treatment with clozapine, which eventually leads to precipitous depletion of PMNs and their precursors. The early appearance of this suppressive substance may offer an early warning for development of agranulocytosis.

Immunological factors responsible for pathogenetic cell degeneration in pregnancy

The placenta has an important role as an immunological barrier during pregnancy. When the placental barrier is disrupted, materno-embryonic transfusion takes place. Several clinical reports relate congenital malformations or abortion to intrauterine bleeding or transplacental transfusion. In an earlier experiment, pathogenetic cell degeneration was induced using an in vitro whole rat embryo culture. Transplacental transfusion was simulated by intracardiac injection of an allogeneic rat-antirat serum directed against the blood group antigens. The present study examines the morphological and immunological effects on the development of rat embryos 9 to 10 days old (stages 8-10 somites) of the separate administration of primary allogeneic antisera, obtained 10-17 days after immunization, and secondary allogeneic antisera, obtained after booster immunization on day 45-52. Rat-antirat alloantibodies were directed against the blood group antigens. Transplacental transfusion was simulated by the embryonic intracardiac microinjection of approximately 0.5 microliters serum enriched with either primary or secondary obtained allogeneic antibodies. After 48 hours' incubation, the embryos were examined microscopically, and it appeared that the secondary antisera, which had hemolytic activity, was more potent (P less than 0.005) in the induction of pathogenetic cell degeneration. It is well known that IgG antibodies display hemolytic activity. This finding was confirmed by direct immunofluorescence performed on rat embryos 2, 4, and 6 hours after injection, where incubation with rabbit-antirat anti-IgG antibodies gave a strong reaction. The hypothesis discussed is whether or not pathogenetic cell degeneration subsequent to transplacental transfusion of maternal antibodies can be initiated by similar immunological events.

A correlation between serum immunosuppressive acidic protein and altered immunocompetence in patients with brain tumours

The present investigation was conducted to document a correlation between the serum levels of immunosuppressive acidic protein (IAP) and depressed lymphocyte responsiveness to mitogens in vitro in patients with intracranial tumours, and to delineate the possible roles of IAP upon immunocompetence in these patients. It was thought that high concentrations of IAP present in the serum of brain-tumour patients may play a significant role in the immunosuppression seen in this patient population. The effect of IAP upon mitogen-stimulated lymphocyte function was evaluated by tritiated (3H)-thymidine incorporation. Lymphocytes from both 30 patients with intracranial tumours and 30 normal individuals were incubated for 90 hours in culture medium in the presence of three mitogens: phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). Lymphocytes obtained from patients with brain tumours and cultured in autologous serum displayed a significant depression of 3H-thymidine incorporation, as was observed in previous studies. In addition, a significant suppression of mitogen-induced activation of the normal lymphocytes was demonstrated in the presence of allogeneic patients' serum and the percentage of inhibition was found significantly proportional to the IAP concentrations. Furthermore it was also demonstrated that increased levels of serum IAP could significantly correlate with two in vivo aspects of impaired cellular immunity: the decreased lymphocyte counts in the peripheral blood and diminished cutaneous delayed hypersensitivity reactions measured by purified protein derivative (PPD) skin test reactivity. On the other hand, an attempt was also made to investigate changes in humoral immunity and immunoglobulin concentrations were observed not to correspond to the serum IAP levels. These studies suggest a possible connection between serum IAP levels and altered cellular immune competence in brain-tumour patients.

Active and Passive Cutaneous Anaphylaxis in Wbb6f1Mouse, A Mast Cell-Deficient Strain

WBB6F1 mouse, a mast cell-deficient strain, was tested for active and passive cutaneous anaphylactic reactions. Active cutaneous anaphylaxis was not produced in mice which had been immunized for 1 to 2 weeks by an intraperitoneal injection of bovine serum albumin with either adjuvant, Freund's complete adjuvant or Bordetella pertussis organisms, even though circulatory IgE and IgG1 antibodies were raised. Passive cutaneous anaphylaxis (PCA) was also absent, when the mice had been sensitized with allogeneic IgE or IgG1 monoclonal antibodies. However, obvious PCA was produced when allogeneic or xenogeneic hyperimmune serum was employed. These findings indicate that mast cells are not necessarily needed for the production of PCA. Some mechanism quite different from the well-elucidated mechanism, i.e., IgE- or IgG1 antibody-triggered histamine release from mast cells, seems to be operative.