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We studied clinical and immunological outcome of Covid-19 in consecutive CLL patients from a well-defined area during month 1–13 of the pandemic. Sixty patients (median age 71 y, range 43–97) were identified. Median CIRS was eight (4–20). Patients had indolent CLL (n = 38), had completed (n = 12) or ongoing therapy (n = 10). Forty-six patients (77%) were hospitalized due to severe Covid-19 and 11 were admitted to ICU. Severe Covid-19 was equally distributed across subgroups irrespective of age, gender, BMI, CLL status except CIRS (p < 0.05). Fourteen patients (23%) died; age ≥75 y was the only significant risk factor (p < 0.05, multivariate analysis with limited power). Comparing month 1–6 vs 7–13 of the pandemic, deaths were numerically reduced from 32% to 18%, ICU admission from 37% to 15% whereas hospitalizations remained frequent (86% vs 71%). Seroconversion occurred in 33/40 patients (82%) and anti-SARS-CoV-2 antibodies were detectable at six and 12 months in 17/22 and 8/11 patients, respectively. Most (13/17) had neutralizing antibodies and 19/28 had antibodies in saliva. SARS-CoV-2-specific T-cells (ELISpot) were detected in 14/17 patients. Covid-19 continued to result in high admission even among consecutive and young early- stage CLL patients. A robust and durable B and/or T cell immunity was observed in most convalescents., QC 20220504

An emerging field of research is starting to examine the association of infectious pathogens with type 2 diabetes mellitus (T2DM). An understudied parasite of interest is Toxoplasma gondii. Globally, very few studies have been conducted to investigate this association. Additionally, very little data exists on the prevalence of T. gondii in the general Australian population. Our group sought to determine the prevalence, association, and risk factors between T. gondii infection and T2DM from a representative Australian human population. Through a cross-sectional, age-A nd gender-matched case-control study, 150 subjects with T2DM together with 150 control subjects from the Busselton Health Study cohort were investigated. Sera samples were tested for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunosorbent assays. Survey-derived data were also analyzed to evaluate associated risk factors. The IgG seroprevalence was found to be 62% and 66% for the T2DM and control groups, respectively (OR:0.84; p=0.471). IgM antibodies were detected in 5% of the T2DM patients and in 10% of the controls (OR=0.51; p=0.135). There were no significant differences between male and female IgG seroprevalence rates for both groups (OR:0.88, 0.80; p=0.723). The IgG seropositivity rate increased significantly in T2DM patients aged 45-84 years in comparison to those aged 18-44 years (p< 0.05), but this was not observed in the control subjects. No risk factors were associated with T. gondii seropositivity in both groups. The first Australian study of its kind found T. gondii infection in Western Australia to be highly prevalent. The results also showed that there is no serological evidence of an association between T. gondii infection and T2DM in the studied subjects. Australian health authorities should focus on raising awareness of toxoplasma infection and target T. gondii transmission control. Further studies are needed to clarify the role of T. gondii in T2DM. © 20

peer-reviewed, no abstract available, PUBLISHED, peer-reviewed

An emerging field of research is starting to examine the association of infectious pathogens with type 2 diabetes mellitus (T2DM). An understudied parasite of interest is Toxoplasma gondii. Globally, very few studies have been conducted to investigate this association. Additionally, very little data exists on the prevalence of T. gondii in the general Australian population. Our group sought to determine the prevalence, association, and risk factors between T. gondii infection and T2DM from a representative Australian human population. Through a cross-sectional, age-A nd gender-matched case-control study, 150 subjects with T2DM together with 150 control subjects from the Busselton Health Study cohort were investigated. Sera samples were tested for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunosorbent assays. Survey-derived data were also analyzed to evaluate associated risk factors. The IgG seroprevalence was found to be 62% and 66% for the T2DM and control groups, respectively (OR:0.84; p=0.471). IgM antibodies were detected in 5% of the T2DM patients and in 10% of the controls (OR=0.51; p=0.135). There were no significant differences between male and female IgG seroprevalence rates for both groups (OR:0.88, 0.80; p=0.723). The IgG seropositivity rate increased significantly in T2DM patients aged 45-84 years in comparison to those aged 18-44 years (p< 0.05), but this was not observed in the control subjects. No risk factors were associated with T. gondii seropositivity in both groups. The first Australian study of its kind found T. gondii infection in Western Australia to be highly prevalent. The results also showed that there is no serological evidence of an association between T. gondii infection and T2DM in the studied subjects. Australian health authorities should focus on raising awareness of toxoplasma infection and target T. gondii transmission control. Further studies are needed to clarify the role of T. gondii in T2DM. © 20

Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays., Export Date: 9 May 2018; Article; CODEN: MODPE; Correspondence Address: Landegren, U.; Department of Immunology, Genetics and Pathology, Science for Life Laboratory, BMC, Uppsala University, Husargatan 3, Sweden; email: Ulf.Landegren@igp.uu.se; Funding details: VINNOVA; Funding details: 222635; Funding details: 241481; Funding details: NCI, National Cancer Institute; Funding details: #2008:0143, Knut och Alice Wallenbergs Stiftelse; Funding details: FP5, Fifth Framework Programme; Funding details: FP/2007– 2013, FP7, Seventh Framework Programme; Funding details: ERC, European Research Council; Funding details: TRC, The Research Council; Funding details: 294409, ERC, European Research Council; Funding details: IngaBritt och Arne Lundbergs Forskningsstiftelse; Funding details: Uppsala Universitet; Funding text: This work was supported by the Knut and Alice Wallenberg Foundation (#2008:0143), the European Community's 7th Framework Program (FP7/2007–2013) under grant agreement n° 222635 (AffinityProteome) 241481 (Affinomics), The Swedish Research Council, Swedish Governmental Agency for Innovation Systems, IngaBritt and Arne Lundberg Foundation, the European Research Council under the European Union's Seventh Framework Programme (FP/2007– 2013) / ERC Grant Agreement n. 294409 (ProteinSeq), and Uppsala University. UL holds stock in Olink, having rights to the in situ proximity ligation assay technology. We would also like to thank Tara Hiltke at the National Cancer Institute for providing mAbs for in situ proximity ligation assay experiments. QC 20180528

Aim: Case report of renal re-transplantation, reported only once previously. Report: A middle aged recipient received a kidney transplant from a deceased multi-organ donor. After initially doing well, the patient suffered cardiac arrest several days post-operatively and sustained hypoxic brain injury and was declared brain dead. Following the family's consent, the allograft kidney was retrieved and re-transplanted into a man with end-stage renal failure secondary to reflux nephropathy. The lungs were used in a separate recipient but the liver was not transplanted due to suspicion of fatty changes based on macroscopic appearance. Histological analysis of the liver more than 24 hours after transplantation of the other organs revealed coagulative parenchymal necrosis with nuclear inclusions and moderate parenchymal cholestasis, suggestive of herpes viral hepatitis. Examination of the renal implantation biopsy showed histiocytes with enlarged nuclei containing viral inclusions in the capsular fibrous tissue, with positive immunostaining for herpes simplex virus (HSV). Valaciclovir was started immediately after obtaining histological evidence of donor HSV infection and this was subsequently converted to intravenous ganciclovir. Our recipient had pre-formed IgG antibodies to HSV-1 and HSV-2, and was IgM negative pretransplant. HSV viraemia was detected day 5 post-transplant with a viral load of 7688 copies/mL by polymerase chain reaction (PCR) assay. He completed a 30-day course of intravenous ganciclovir before switching to valganciclovir as standard cytomegalovirus prophylaxis. The HSV PCR became undetectable on day 7 of IV ganciclovir and has remained undetectable. The patient remains well with an estimated glomerular filtration rate of 61 mL/min/1.73 m2 and further investigation of the apparent viral transmission is underway. Conclusion(s): We report good short term results of renal re-transplantation and HSV transmission by transplantation.

Aim: Case report of renal re-transplantation, reported only once previously. Report: A middle aged recipient received a kidney transplant from a deceased multi-organ donor. After initially doing well, the patient suffered cardiac arrest several days post-operatively and sustained hypoxic brain injury and was declared brain dead. Following the family's consent, the allograft kidney was retrieved and re-transplanted into a man with end-stage renal failure secondary to reflux nephropathy. The lungs were used in a separate recipient but the liver was not transplanted due to suspicion of fatty changes based on macroscopic appearance. Histological analysis of the liver more than 24 hours after transplantation of the other organs revealed coagulative parenchymal necrosis with nuclear inclusions and moderate parenchymal cholestasis, suggestive of herpes viral hepatitis. Examination of the renal implantation biopsy showed histiocytes with enlarged nuclei containing viral inclusions in the capsular fibrous tissue, with positive immunostaining for herpes simplex virus (HSV). Valaciclovir was started immediately after obtaining histological evidence of donor HSV infection and this was subsequently converted to intravenous ganciclovir. Our recipient had pre-formed IgG antibodies to HSV-1 and HSV-2, and was IgM negative pretransplant. HSV viraemia was detected day 5 post-transplant with a viral load of 7688 copies/mL by polymerase chain reaction (PCR) assay. He completed a 30-day course of intravenous ganciclovir before switching to valganciclovir as standard cytomegalovirus prophylaxis. The HSV PCR became undetectable on day 7 of IV ganciclovir and has remained undetectable. The patient remains well with an estimated glomerular filtration rate of 61 mL/min/1.73 m2 and further investigation of the apparent viral transmission is underway. Conclusion(s): We report good short term results of renal re-transplantation and HSV transmission by transplantation.

HIT is a serious complication and remains a challenge for diagnostic laboratories and clinicians. Only a subset of IgG antibodies to complexes of platelet factor 4 (PF4) and heparin (H) trigger the clinical manifestations of HIT by activating platelets, and these can only be identified with platelet activation/aggregation (functional) assays. The 14C-serotonin release assay (SRA), the gold standard in HIT diagnosis, is highly sensitive but only performed in one Australian laboratory and hence unsuitable for routine use. We have developed a whole blood impedance aggregometry (WBIA-Multiplate) and in a small cohort of HIT-antibody positive patients have shown it to be superior to light transmission aggregometry and as sensitive as the SRA. We obtained ethics approval to conduct a multi-centre study to validate the WBIA as a suitable diagnostic tool in HIT. We have tested 158 samples positive for H-PF4 antibodies by PaGIA or ELISA. Using a selected donor (high responder), 65 samples were positive by WBIA (aggregation with low dose 0.5 IU/mL H but not with high dose). SRA was performed using the same donor platelets, serotonin was released from 80 samples (> 20% release with 0.1 IU/ mL H) with only 70 true HIT samples (release < 20% with 100 IU/ mL H). Eleven samples exhibited discrepant results: one strongly positive (89% release) and seven weakly positive by SRA (average release 40%, range 22-67%) were WBIA negative. These eight samples were retested using a random donor, only three remained SRA positive. Three samples were SRA negative but strongly WBIA positive. These discrepant cases will be analysed for clinical outcomes and potential interfering substances. In this multi-centre study, with a selected donor, WBIA had a sensitivity of 88.6% which could rise to 95% using random donors for SRA, a specificity of 96.6% and a PPV of 95.4%. WBIA is easy to perform with rapid turn-around time and warrants further investigation as confirmatory assay for platelet-activating

HIT is a serious complication and remains a challenge for diagnostic laboratories and clinicians. Only a subset of IgG antibodies to complexes of platelet factor 4 (PF4) and heparin (H) trigger the clinical manifestations of HIT by activating platelets, and these can only be identified with platelet activation/aggregation (functional) assays. The 14C-serotonin release assay (SRA), the gold standard in HIT diagnosis, is highly sensitive but only performed in one Australian laboratory and hence unsuitable for routine use. We have developed a whole blood impedance aggregometry (WBIA-Multiplate) and in a small cohort of HIT-antibody positive patients have shown it to be superior to light transmission aggregometry and as sensitive as the SRA. We obtained ethics approval to conduct a multi-centre study to validate the WBIA as a suitable diagnostic tool in HIT. We have tested 158 samples positive for H-PF4 antibodies by PaGIA or ELISA. Using a selected donor (high responder), 65 samples were positive by WBIA (aggregation with low dose 0.5 IU/mL H but not with high dose). SRA was performed using the same donor platelets, serotonin was released from 80 samples (> 20% release with 0.1 IU/ mL H) with only 70 true HIT samples (release < 20% with 100 IU/ mL H). Eleven samples exhibited discrepant results: one strongly positive (89% release) and seven weakly positive by SRA (average release 40%, range 22-67%) were WBIA negative. These eight samples were retested using a random donor, only three remained SRA positive. Three samples were SRA negative but strongly WBIA positive. These discrepant cases will be analysed for clinical outcomes and potential interfering substances. In this multi-centre study, with a selected donor, WBIA had a sensitivity of 88.6% which could rise to 95% using random donors for SRA, a specificity of 96.6% and a PPV of 95.4%. WBIA is easy to perform with rapid turn-around time and warrants further investigation as confirmatory assay for platelet-activating

The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization. © 2009 Elsevier B.V. All rights reserved.

The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization. © 2009 Elsevier B.V. All rights reserved.

Semen samples from infertile men were assessed for sperm autoimmunity by direct immunobead assay for immunoglobulin (Ig)A and IgG sperm antibodies and mucus penetration test. Immunosuppressive activity in seminal plasma was measured by an in-vitro bioassay employing dose-dependent inhibition of phytohaemagglutinin-induced activation of rat thymocytes, in the presence or absence of hydroxylamine (0.1 mM), an inhibitor of polyamine oxidation. All seminal plasma samples, regardless of autoimmune status, caused inhibition of T-lymphocyte activation, and hydroxylamine reduced this bioactivity by ~ 50%. Dialysis (<3500 molecular weight) also significantly reduced seminal plasma bioactivity, both in the presence and absence of hydroxylamine. In the presence of hydroxylamine, there was a negative correlation between IgA, but not IgG, antibody concentrations and lymphosuppressive activity in seminal plasma. Antibody-positive samples displaying impaired sperm function, as indicated by the mucus penetration test, had reduced activity compared with other samples. In contrast, there was no relationship between sperm autoimmunity and lymphosuppressive activity assayed in the absence of hydroxylamine. The data indicate that T-lymphocyte inhibition by human seminal plasma is due to multiple factors, and reduced amounts of these factors may contribute to the development and/or persistence of sperm autoimmunity in infertile men; however, differences in polyamine substrates available for oxidation in semen do not appear to be a major contributing factor.

Semen samples from infertile men were assessed for sperm autoimmunity by direct immunobead assay for immunoglobulin (Ig)A and IgG sperm antibodies and mucus penetration test. Immunosuppressive activity in seminal plasma was measured by an in-vitro bioassay employing dose-dependent inhibition of phytohaemagglutinin-induced activation of rat thymocytes, in the presence or absence of hydroxylamine (0.1 mM), an inhibitor of polyamine oxidation. All seminal plasma samples, regardless of autoimmune status, caused inhibition of T-lymphocyte activation, and hydroxylamine reduced this bioactivity by ~ 50%. Dialysis (<3500 molecular weight) also significantly reduced seminal plasma bioactivity, both in the presence and absence of hydroxylamine. In the presence of hydroxylamine, there was a negative correlation between IgA, but not IgG, antibody concentrations and lymphosuppressive activity in seminal plasma. Antibody-positive samples displaying impaired sperm function, as indicated by the mucus penetration test, had reduced activity compared with other samples. In contrast, there was no relationship between sperm autoimmunity and lymphosuppressive activity assayed in the absence of hydroxylamine. The data indicate that T-lymphocyte inhibition by human seminal plasma is due to multiple factors, and reduced amounts of these factors may contribute to the development and/or persistence of sperm autoimmunity in infertile men; however, differences in polyamine substrates available for oxidation in semen do not appear to be a major contributing factor.

We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three- layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.

We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three- layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.

Cystic fibrosis (CF) patients require pancreatic enzyme replacement therapy to correct pancreatic insufficiency. These enzymes are derived from porcine pancreas and are known to be antigenic. To determine the possible clinical consequences, a specific ELISA was developed to detect IgG antibody directed against porcine trypsin (PTAb) in the sera of CF patients. The assay was used to evaluate the occurrence of PTAb in a cross sectional study of 103 CF patients in relation to the introduction of porcine enzyme therapy, clinical status and genotype. Antibodies against porcine trypsin were detected in the sera of 63% of patients unrelated to the age of commencement or the duration of enzyme therapy. No differences were observed in the clinical status of CF patients who had developed PTAb (n = 65) and those who had no detectable PTAb (n = 38) as determined from: the current prescribed dose of porcine pancreatic enzyme capsules; Z scores for height and weight; and respiratory function tests. It is suggested that the PTAb commonly found in the sera of CF patients are of doubtful clinical significance but the prospect of PTAb contributing to immune complex disease should be examined further.

Nosaka, K., Hunter, M., Zhang, J., Meng, X., Song, M., & Wang, W. (2020). First age-and gender-matched case-control study in Australia examining the possible association between toxoplasma gondii Infection and type 2 diabetes mellitus: The Busselton Health Study. Journal of Parasitology Research, 2020, Article 3142918. https://doi.org/10.1155/2020/3142918